Optimized lentiviral transfer vectors and uses thereof

ABSTRACT

The invention features lentiviral transfer vectors that include heterologous nucleic acids to be introduced into a cell. The lentiviral transfer vector may be characterized by the following features: (a) including a cytomegalovirus (CMV) promoter; (b) including a polynucleotide encoding a partial gag protein that includes a mutated INS1 inhibitory sequence that reduces restriction of nuclear export of RNA; (c) not including a polynucleotide encoding the INS2, INS3, and INS4 inhibitory sequences of gag; (d) not including an SV40 origin of replication and/or an f1 origin of replication; (e) including a cPPT sequence that contains splice site; (f) including an EF1alpha promoter with intact splice donor and acceptor sites; and (g) including hepatitis B PRE with mutation in start codon of X protein ORF.

FIELD OF THE INVENTION

The present invention relates to lentiviral transfer vectors and methodsof using such vectors.

BACKGROUND OF THE INVENTION

Expression of heterologous genes in cells is desirable for manytherapeutically relevant applications. One method of introducing aheterologous gene into a cell involves the use of transfer vectorswhich, when transfected into a host cell, induce the host cell toproduce viral particles including the heterologous gene. The viralparticles can then be used to infect a target cell, thereby inducing thetarget cell to express the heterologous gene. Viruses useful in suchmethods include lentiviruses, adenoviruses, and adeno-associatedviruses. Some existing lentiviral vectors exhibit low gene expressionlevels and may be extremely large. In addition, there can be biosafetyand toxicity concerns with respect to such vectors. As such,transfection and viral production using such vectors may be slow,inefficient, laborious, and expensive. Thus, a need exists for improvedlentiviral vectors suitable for rapid and efficient production ofviruses that are useful for inducing heterologous gene expression intarget cells.

SUMMARY OF THE INVENTION

In a first aspect, the invention provides lentiviral transfer vectors,optionally including one or more heterologous nucleic acid sequences(which are optionally downstream from a Kozak sequence), and beingcharacterized by at least two of the following features: (a) including acytomegalovirus (CMV) promoter, (b) including a polynucleotide encodingat least a portion of a gag protein that includes a mutated INS1inhibitory sequence that reduces restriction of nuclear export of RNArelative to wild-type INS1, (c) not including a polynucleotide encodingthe INS2, INS3, and INS4 inhibitory sequences of gag, and (d) notincluding an SV40 origin of replication and/or an f1 origin ofreplication. In various embodiments, lentiviral transfer vector ischaracterized by at least three or all four of features (a)-(d).

In various examples, the lentiviral transfer includes a polynucleotideencoding a 150-250 (e.g., 168) nucleotide portion of a gag protein that(i) includes a mutated INS1 inhibitory sequence that reduces restrictionof nuclear export of RNA relative to wild-type INS1, (ii) contains twonucleotide insertion that results in frame shift and prematuretermination, and/or (iii) does not include INS2, INS3, and INS4inhibitory sequences of gag.

In additional examples, the lentiviral transfer vector further includesone or more elements selected from the group consisting of a packagingsignal (psi), a partial gag sequence adjacent to or partiallyoverlapping with psi, a rev-response element, a partial env sequence,and a cPPT sequence from pol, the sequences of which optionallyoriginate from HIV-1 isolate NL4-3 or SF3. In various examples, the cPPTsequence includes about 150-250 (e.g., 178-181) nucleotides and includessplice acceptor SA1 sequence.

The lentiviral transfer vectors can optionally include one or morerestriction sites positioned between elements of the vector (see, e.g.,Tables 3-5 below for exemplary locations).

Further, the lentiviral transfer vectors can optionally include apost-transcriptional regulatory element (PRE). For example, a woodchuckhepatitis virus PRE (WPRE; having, e.g., at least 95%, 96%, 97%, 98%,99%, or 100% sequence identity to SEQ ID NO: 78), or a hepatitis B virusisolate bba6 PRE (HPRE; having, e.g., at least 95%, 96%, 97%, 98%, 99%,or 100% sequence identity to SEQ ID NO: 79), and optionally wherein theHPRE including an inactivating mutation in an X protein-encodingsequence. The lentiviral transfer vectors further can include asubgenomic promoter that can be used to drive expression of a transgene.In one example, the promoter is an EF1a promoter (e.g., an EF1a promoterhaving a nucleic acid sequence having at least 95%, 96%, 97%, 98%, 99%,or 100% identity to SEQ ID NO: 71). The EF1a promoter optionally is fulllength and includes intact splice donor and splice acceptor sequences(SEQ ID NOs:72 and 73, respectively) (see, e.g., SEQ ID NO:95).

The lentiviral components of the lentiviral transfer vectors canoptionally originate from HIV-1 (e.g., HIV-1 strain NL4-3 or SF3). Invarious embodiments, in the lentiviral transfer vectors, the sequenceencoding the partial gag protein has less than 90% sequence identity toa corresponding region of gag protein encoded by a packaging plasmidused with the lentiviral transfer vector (e.g., pMDLgpRRE packagingplasmid).

In another aspect, the invention includes lentiviral transfer vectorsthat include one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, or 22) of the following features: (i) aCMV promoter including a nucleic acid sequence having at least 95%identity to SEQ ID NO: 52, (ii) an LTR R region including a nucleic acidsequence having at least 95% identity to SEQ ID NO: 53, (iii) an LTR U5region including a nucleic acid sequence having at least 95% identity toSEQ ID NO: 54, (iv) a primer binding site including a nucleic acidsequence having at least 95% identity to SEQ ID NO: 55, (v) a packagingsignal including a nucleic acid sequence having at least 95% identity toSEQ ID NO: 56, (vi) a major splice donor site including a nucleic acidsequence having at least 95% identity to SEQ ID NO: 57, which is withinthe packaging signal, (vii) a partial gag sequence including a nucleicacid sequence having at least 95% identity to SEQ ID NO:58, (viii) apartial env sequence including a nucleic acid sequence having at least95% identity to SEQ ID NO:60, (ix) a rev-response element including anucleic acid sequence having at least 95% identity to SEQ ID NO: 62, (x)a partial env sequence including a nucleic acid sequence having at least95% identity to SEQ ID NO:64, (xi) a splice acceptor site including anucleic acid sequence having at least 95% identity to SEQ ID NO: 65,which is within the partial env sequence of part (x), (xii) a centralpolypurine tract including a nucleic acid sequence having at least 95%identity to SEQ ID NO: 67, 92, or 93, (xiii) a splice acceptor siteincluding a nucleic acid sequence having at least 95% identity to SEQ IDNO: 68 or 94, which is within the central polypurine tract, (xiv) anEF1alpha promoter having at least 95% sequence identity to SEQ ID NO:71or 95, (xv) a constitutive splice donor (CD) site including a nucleicacid sequence having at least 95% identity to SEQ ID NO: 72, which iswithin the EF1alpha promoter, (xvi) a constitutive splice acceptor (CA)site including a nucleic acid sequence having at least 95% identity toSEQ ID NO: 73, which is within the EF1alpha promoter, (xvii) apolynucleotide encoding an EGFP including a nucleic acid sequence havingat least 95% identity to SEQ ID NO: 76 and/or a transgene sequence,(xviii) a PRE sequence including a nucleic acid sequence having at least95% sequence identity to SEQ ID NO: 78 or 79, (xix) a partial nefsequence including a nucleic acid sequence having at least 95% sequenceidentity to SEQ ID NO:83, (xx) a dU3 sequence including a nucleic acidsequencing having at least 95% sequence identity to SEQ ID NO:84, (xxi)an LTR R region including a nucleic acid sequence having at least 95%identity to SEQ ID NO: 85, and (xxii) an LTR U5 region including anucleic acid sequence having at least 95% identity to SEQ ID NO: 86. Thesequence identities of these components can optionally be 96%, 97%, 98%,99%, or 100%. Examples of combinations include those containing: i, vii,viii, ix, x, xii, xiv, and xviii; wherein x optionally includes xi, xiioptionally includes xiii, and xiv optionally includes xv and xvi. Inaddition, the combination may also include ii, iii, iv, v, xix, xx, xxi,and/or xxv, optionally wherein v includes vi. Further, any of thesecombinations may also include xvii.

The lentiviral transfer vectors of the invention can optionally includea heterologous nucleic acid sequence that encodes a protein, e.g., EGFPand or a chimeric antigen receptor (CAR). In the case of a CAR, the CARcan optionally include, in a N-terminal to C-terminal direction, anantigen binding domain (e.g., an scFv), a transmembrane domain, and oneor more signaling domains (e.g., one or more primary signaling domains(e.g., a CD3-zeta stimulatory domain) and/or one or more costimulatorysignaling domains (e.g., an intracellular domain selected from acostimulatory protein selected from the group consisting of CD27, CD28,4-1 BB (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, ICAM-1,lymphocyte function-associated antigen-1 (LFA-1), CD2, CDS, CD7, CD287,LIGHT, NKG2C, NKG2D, SLAMF7, NKp80, NKp30, NKp44, NKp46, CD160, B7-H3,and a ligand that specifically binds with CD83)).

In various examples, the antigen binding domain binds to an antigenselected from the group consisting of CD19; CD123; CD22; CD30; CD171;CS-1; C-type lectin-like molecule-1, CD33; epidermal growth factorreceptor variant III (EGFRvIII); ganglioside G2 (GD2); ganglioside GD3;TNF receptor family member B cell maturation (BCMA); Tn antigen ((Tn Ag)or (GaINAcα-Ser/Thr)); prostate-specific membrane antigen (PSMA);Receptor tyrosine kinase-like orphan receptor 1 (ROR1); Fms-LikeTyrosine Kinase 3 (FLT3); Tumor-associated glycoprotein 72 (TAG72);CD38; CD44v6; Carcinoembryonic antigen (CEA); Epithelial cell adhesionmolecule (EPCAM); B7H3 (CD276); KIT (CD117); Interleukin-13 receptorsubunit alpha-2; mesothelin; Interleukin 11 receptor alpha (IL-11 Ra);prostate stem cell antigen (PSCA); Protease Serine 21; vascularendothelial growth factor receptor 2 (VEGFR2); Lewis(Y) antigen; CD24;Platelet-derived growth factor receptor beta (PDGFR-beta);Stage-specific embryonic antigen-4 (SSEA-4); CD20; Folate receptoralpha; Receptor tyrosine-protein kinase ERBB2 (Her2/neu); Mucin 1, cellsurface associated (MUC1); epidermal growth factor receptor (EGFR);neural cell adhesion molecule (NCAM); Prostase; prostatic acidphosphatase (PAP); elongation factor 2 mutated (ELF2M); Ephrin B2;fibroblast activation protein alpha (FAP); insulin-like growth factor 1receptor (IGF-I receptor), carbonic anhydrase IX (CAIX); Proteasome(Prosome, Macropain) Subunit, Beta Type, 9 (LMP2); glycoprotein 100(gp100); oncogene fusion protein consisting of breakpoint cluster region(BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl)(bcr-abl); tyrosinase; ephrin type-A receptor 2 (EphA2); Fucosyl GM1;sialyl Lewis adhesion molecule (sLe); ganglioside GM3; transglutaminase5 (TGS5); high molecular weight-melanoma-associated antigen (HMWMAA);o-acetyl-G D2 ganglioside (OAcGD2); Folate receptor beta; tumorendothelial marker 1 (TEM1/CD248); tumor endothelial marker 7-related(TEM7R); claudin 6 (CLDN6); thyroid stimulating hormone receptor (TSHR);G protein-coupled receptor class C group 5, member D (GPRC5D);chromosome X open reading frame 61 (CXORF61); CD97; CD179a; anaplasticlymphoma kinase (ALK); Polysialic acid; placenta-specific 1 (PLAC1);hexasaccharide portion of globoH glycoceramide (GloboH); mammary glanddifferentiation antigen (NY-BR-1); uroplakin 2 (UPK2); Hepatitis A viruscellular receptor 1 (HAVCR1); adrenoceptor beta 3 (ADRB3); pannexin 3(PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6complex, locus K 9 (LY6K); Olfactory receptor 51E2 (OR51E2); TCR GammaAlternate Reading Frame Protein (TARP); Wilms tumor protein (WT1);Cancer/testis antigen 1 (NY-ESO-1); Cancer/testis antigen 2 (LAGE-1a);Melanoma-associated antigen 1 (MAGE-A1); ETS translocation-variant gene6, located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); XAntigen Family, Member 1A (XAGE1); angiopoietin-binding cell surfacereceptor 2 (Tie 2); melanoma cancer testis antigen-1 (MAD-CT-1);melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1;tumor protein p53 (p53); p53 mutant; prostein; surviving; telomerase;prostate carcinoma tumor antigen-1, melanoma antigen recognized by Tcells 1; Rat sarcoma (Ras) mutant; human Telomerase reversetranscriptase (hTERT); sarcoma translocation breakpoints; melanomainhibitor of apoptosis (ML-IAP); ERG (transmembrane protease, serine 2(TMPRSS2) ETS fusion gene); N-Acetyl glucosaminyl-transferase V (NA17);paired box protein Pax-3 (PAX3); Androgen receptor; Cyclin B1; v-mycavian myelocytomatosis viral oncogene neuroblastoma derived homolog(MYCN); Ras Homolog Family Member C (RhoC); Tyrosinase-related protein 2(TRP-2); Cytochrome P450 1 B1 (CYP1B1); CCCTC-Binding Factor (ZincFinger Protein)-Like, Squamous Cell Carcinoma Antigen Recognized By TCells 3 (SART3); Paired box protein Pax-5 (PAX5); proacrosin bindingprotein sp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase(LCK); A kinase anchor protein 4 (AKAP-4); synovial sarcoma, Xbreakpoint 2 (SSX2); Receptor for Advanced Glycation Endproducts(RAGE-1); renal ubiquitous 1 (RU1); renal ubiquitous 2 (RU2); legumain;human papilloma virus E6 (HPV E6); human papilloma virus E7 (HPV E7);intestinal carboxyl esterase; heat shock protein 70-2 mutated (muthsp70-2); CD79a; CD79b; CD72; Leukocyte-associated immunoglobulin-likereceptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR or CD89);Leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2);CD300 molecule-like family member f (CD300LF); C-type lectin domainfamily 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2);EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2);lymphocyte antigen 75 (LY75); Glypican-3 (GPC3); Fc receptor-like 5(FCRL5); and immunoglobulin lambda-like polypeptide 1 (IGLL1).

In a specific example, the CAR includes an anti-CD19 antibody or afragment thereof, a 4-1 BB (CD137) transmembrane domain, and a CD3-zetasignaling domain.

In another aspect, the invention includes lentiviral transfer vectorsincluding, from 5′ to 3′, one or more (e.g., all) of the followingelements in operable association: a promoter, a packaging signal (psi)including a major splice donor site (SD), a partial gag sequence, apartial env sequence, a Rev-response element (RRE), a partial envsequence including splice acceptor site (SA7), a central polypurinetract (cPPT) including a splice acceptor site (SA1), an EF1a promoter,which comprises a constitutive splice donor site (CD) and a constitutivesplice acceptor site (CD), optionally a gene encoding EGFP and/or aheterologous nucleic acid sequence, and a post-transcriptionalregulatory element.

In one example, the invention includes lentiviral transfer vectorincluding, from 5′ to 3′, one or more of the following elements inoperable association: a CMV promoter, an LTR R region, an LTR U5 region,a primer binding site (PBS), a packaging signal (psi) including a majorsplice donor site (SD), a partial gag sequence, a partial env sequence,a Rev-response element (RRE), a partial env sequence including spliceacceptor site (SA7), a central polypurine tract (cPPT) including asplice acceptor site (SA1), an EF1a promoter, optionally a gene encodingEGFP and/or a heterologous nucleic acid sequence, a post-transcriptionalregulatory element, an LTR R region, an LTR U5 region, an SV40 polyAtail, a kanamycin resistance gene (nptII), and a pUC origin ofreplication.

In various examples, the post-transcriptional regulatory element is awoodchuck hepatitis virus PRE (WPRE) or a hepatitis B virus isolate bba6PRE (HPRE), as described herein.

In further examples, the heterologous nucleic acid sequence encodes achimeric antigen receptor (CAR), as described herein. In one example,the CAR can include an anti-CD19 antibody or a fragment thereof, a 4-1BB (CD137) transmembrane domain, and a CD3-zeta signaling domain.

In another aspect, the invention includes a host cell (e.g., a 293Tcell, a Jurkat T cell, or a primary human T cell) including a lentiviraltransfer vector as described herein. Optionally, the host call canfurther include one or more lentiviral packaging vectors.

In a further aspect, the invention includes compositions including alentiviral transfer vector as described herein and one or more packagingvectors.

In an additional aspect, the invention includes methods of producing alentivirus capable of expressing a heterologous nucleic acid sequence.These methods can optionally include (a) introducing into a cell (e.g.,a 293T cell, a Jurkat T cell, or a primary human T cell): (i) alentiviral transfer vector as described herein, and (ii) one or morelentiviral packaging vectors; and (b) expressing viral proteins encodedby the lentiviral transfer vector and/or the packaging vector in thecell, thereby producing a lentivirus including the heterologous nucleicacid sequence of the lentiviral transfer vector.

Definitions

The term “Chimeric Antigen Receptor” or alternatively a “CAR” refers toa set of polypeptides, typically two in the simplest embodiments, whichwhen in an immune effector cell, provides the cell with specificity fora target cell, typically a cancer cell, and with intracellular signalgeneration. In some embodiments, a CAR comprises at least anextracellular antigen binding domain, a transmembrane domain and acytoplasmic signaling domain (also referred to herein as “anintracellular signaling domain”) comprising a functional signalingdomain derived from a stimulatory molecule and/or costimulatory moleculeas defined below. In some aspects, the set of polypeptides arecontiguous with each other. In some embodiments, the set of polypeptidesinclude a dimerization switch that, upon the presence of a dimerizationmolecule, can couple the polypeptides to one another, e.g., can couplean antigen binding domain to an intracellular signaling domain. In oneaspect, the stimulatory molecule is the zeta chain associated with the Tcell receptor complex. In one aspect, the cytoplasmic signaling domainfurther comprises one or more functional signaling domains derived fromat least one costimulatory molecule as defined below. In one aspect, thecostimulatory molecule is chosen from the costimulatory moleculesdescribed herein, e.g., 4-1 BB (i.e., CD137), CD27 and/or CD28. In oneaspect, the CAR comprises a chimeric fusion protein comprising anextracellular antigen binding domain, a transmembrane domain and anintracellular signaling domain comprising a functional signaling domainderived from a stimulatory molecule. In one aspect, the CAR comprises achimeric fusion protein comprising an extracellular antigen bindingdomain, a transmembrane domain and an intracellular signaling domaincomprising a functional signaling domain derived from a costimulatorymolecule and a functional signaling domain derived from a stimulatorymolecule. In one aspect, the CAR comprises a chimeric fusion proteincomprising an extracellular antigen binding domain, a transmembranedomain and an intracellular signaling domain comprising two functionalsignaling domains derived from one or more costimulatory molecule(s) anda functional signaling domain derived from a stimulatory molecule. Inone aspect, the CAR comprises a chimeric fusion protein comprising anextracellular antigen binding domain, a transmembrane domain and anintracellular signaling domain comprising at least two functionalsignaling domains derived from one or more costimulatory molecule(s) anda functional signaling domain derived from a stimulatory molecule. Inone aspect the CAR comprises an optional leader sequence at theamino-terminus (N-ter) of the CAR fusion protein. In one aspect, the CARfurther comprises a leader sequence at the N-terminus of theextracellular antigen binding domain, wherein the leader sequence isoptionally cleaved from the antigen binding domain (e.g., a scFv) duringcellular processing and localization of the CAR to the cellularmembrane.

A CAR that comprises an antigen binding domain (e.g., a scFv, or TCR)that targets a specific tumor maker X, such as those described herein,is also referred to as XCAR. For example, a CAR that comprises anantigen binding domain that targets CD19 is referred to as CD19CAR.

The term “signaling domain” refers to the functional portion of aprotein which acts by transmitting information within the cell toregulate cellular activity via defined signaling pathways by generatingsecond messengers or functioning as effectors by responding to suchmessengers.

The term “antibody,” as used herein, refers to a protein, or polypeptidesequence derived from an immunoglobulin molecule which specificallybinds with an antigen. Antibodies can be polyclonal or monoclonal,multiple or single chain, or intact immunoglobulins, and may be derivedfrom natural sources or from recombinant sources. Antibodies can betetramers of immunoglobulin molecules.

The term “antibody fragment” refers to at least one portion of anantibody, that retains the ability to specifically interact with (e.g.,by binding, steric hinderance, stabilizing/destabilizing, spatialdistribution) an epitope of an antigen. Examples of antibody fragmentsinclude, but are not limited to, Fab, Fab′, F(ab′)₂, Fv fragments, scFvantibody fragments, disulfide-linked Fvs (sdFv), a Fd fragmentconsisting of the VH and CH1 domains, linear antibodies, single domainantibodies such as sdAb (either VL or VH), camelid VHH domains,multi-specific antibodies formed from antibody fragments such as abivalent fragment comprising two Fab fragments linked by a disulfidebridge at the hinge region, and an isolated CDR or other epitope bindingfragments of an antibody. An antigen binding fragment can also beincorporated into single domain antibodies, maxibodies, minibodies,nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR andbis-scFv (see, e.g., Hollinger and Hudson, Nature Biotechnology23:1126-1136, 2005). Antigen binding fragments can also be grafted intoscaffolds based on polypeptides such as a fibronectin type III (Fn3)(seeU.S. Pat. No. 6,703,199, which describes fibronectin polypeptideminibodies).

The term “scFv” refers to a fusion protein comprising at least oneantibody fragment comprising a variable region of a light chain and atleast one antibody fragment comprising a variable region of a heavychain, wherein the light and heavy chain variable regions arecontiguously linked, e.g., via a synthetic linker, e.g., a shortflexible polypeptide linker, and capable of being expressed as a singlechain polypeptide, and wherein the scFv retains the specificity of theintact antibody from which it is derived. Unless specified, as usedherein an scFv may have the VL and VH variable regions in either order,e.g., with respect to the N-terminal and C-terminal ends of thepolypeptide, the scFv may comprise VL-linker-VH or may compriseVH-linker-VL.

The portion of the CAR of the invention comprising an antibody orantibody fragment thereof may exist in a variety of forms where theantigen binding domain is expressed as part of a contiguous polypeptidechain including, for example, a single domain antibody fragment (sdAb),a single chain antibody (scFv), a humanized antibody or bispecificantibody (Harlow et al., 1999, In: Using Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989,In: Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Houstonet al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al.,1988, Science 242:423-426). In one aspect, the antigen binding domain ofa CAR composition of the invention comprises an antibody fragment. In afurther aspect, the CAR comprises an antibody fragment that comprises ascFv. The precise amino acid sequence boundaries of a given CDR can bedetermined using any of a number of well-known schemes, including thosedescribed by Kabat et al. (1991), “Sequences of Proteins ofImmunological Interest,” 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (“Kabat” numbering scheme),Al-Lazikani et al., (1997) JMB 273,927-948 (“Chothia” numbering scheme),or a combination thereof.

As used herein, the term “binding domain” or “antibody molecule” refersto a protein, e.g., an immunoglobulin chain or fragment thereof,comprising at least one immunoglobulin variable domain sequence. Theterm “binding domain” or “antibody molecule” encompasses antibodies andantibody fragments. In an embodiment, an antibody molecule is amultispecific antibody molecule, e.g., it comprises a plurality ofimmunoglobulin variable domain sequences, wherein a first immunoglobulinvariable domain sequence of the plurality has binding specificity for afirst epitope and a second immunoglobulin variable domain sequence ofthe plurality has binding specificity for a second epitope. In anembodiment, a multispecific antibody molecule is a bispecific antibodymolecule. A bispecific antibody has specificity for no more than twoantigens. A bispecific antibody molecule is characterized by a firstimmunoglobulin variable domain sequence which has binding specificityfor a first epitope and a second immunoglobulin variable domain sequencethat has binding specificity for a second epitope.

The portion of the CAR of the invention comprising an antibody orantibody fragment thereof may exist in a variety of forms where theantigen binding domain is expressed as part of a contiguous polypeptidechain including, for example, a single domain antibody fragment (sdAb),a single chain antibody (scFv), a humanized antibody, or bispecificantibody (Harlow et al., 1999, In: Using Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989,In: Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Houstonet al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al.,1988, Science 242:423-426). In one aspect, the antigen binding domain ofa CAR composition of the invention comprises an antibody fragment. In afurther aspect, the CAR comprises an antibody fragment that comprises ascFv.

The term “antibody heavy chain,” refers to the larger of the two typesof polypeptide chains present in antibody molecules in their naturallyoccurring conformations, and which normally determines the class towhich the antibody belongs.

The term “antibody light chain,” refers to the smaller of the two typesof polypeptide chains present in antibody molecules in their naturallyoccurring conformations. Kappa (K) and lambda (A) light chains refer tothe two major antibody light chain isotypes.

The term “recombinant antibody” refers to an antibody which is generatedusing recombinant DNA technology, such as, for example, an antibodyexpressed by a bacteriophage or yeast expression system. The term shouldalso be construed to mean an antibody which has been generated by thesynthesis of a DNA molecule encoding the antibody and which DNA moleculeexpresses an antibody protein, or an amino acid sequence specifying theantibody, wherein the DNA or amino acid sequence has been obtained usingrecombinant DNA or amino acid sequence technology which is available andwell known in the art.

The term “antigen” or “Ag” refers to a molecule that provokes an immuneresponse. This immune response may involve either antibody production,or the activation of specific immunologically-competent cells, or both.The skilled artisan will understand that any macromolecule, includingvirtually all proteins or peptides, can serve as an antigen.Furthermore, antigens can be derived from recombinant or genomic DNA. Askilled artisan will understand that any DNA, which comprises anucleotide sequences or a partial nucleotide sequence encoding a proteinthat elicits an immune response therefore encodes an “antigen” as thatterm is used herein. Furthermore, one skilled in the art will understandthat an antigen need not be encoded solely by a full length nucleotidesequence of a gene. It is readily apparent that the present inventionincludes, but is not limited to, the use of partial nucleotide sequencesof more than one gene and that these nucleotide sequences are arrangedin various combinations to encode polypeptides that elicit the desiredimmune response. Moreover, a skilled artisan will understand that anantigen need not be encoded by a “gene” at all. It is readily apparentthat an antigen can be generated synthesized or can be derived from abiological sample, or might be macromolecule besides a polypeptide. Sucha biological sample can include, but is not limited to a tissue sample,a tumor sample, a cell or a fluid with other biological components.

The term “autologous” refers to any material derived from the sameindividual (e.g., a cell or organism) to whom it is later to bere-introduced into the individual. The term “heterologous” may refer toany material derived from a different individual (e.g., a cell ororganism) than the individual to whom the material is introduced. Insome instances, the term “heterologous” may refer to any materialderived from an individual (e.g., a cell or organism) of a differentspecies than the individual to whom the material is introduced.

An “intracellular signaling domain,” as the term is used herein, refersto an intracellular portion of a molecule. The intracellular signalingdomain generates a signal that promotes an immune effector function ofthe CAR containing cell, e.g., a CART cell. Examples of immune effectorfunction, e.g., in a CART cell, include cytolytic activity and helperactivity, including the secretion of cytokines.

In an embodiment, the intracellular signaling domain can comprise aprimary intracellular signaling domain. Exemplary primary intracellularsignaling domains include those derived from the molecules responsiblefor primary stimulation, or antigen dependent simulation. In anembodiment, the intracellular signaling domain can comprise acostimulatory intracellular domain. Exemplary costimulatoryintracellular signaling domains include those derived from moleculesresponsible for costimulatory signals, or antigen independentstimulation. For example, in the case of a CART, a primary intracellularsignaling domain can comprise a cytoplasmic sequence of a T cellreceptor, and a costimulatory intracellular signaling domain cancomprise cytoplasmic sequence from co-receptor or costimulatorymolecule.

A primary intracellular signaling domain can comprise a signaling motifwhich is known as an immunoreceptor tyrosine-based activation motif orITAM. Examples of ITAM containing primary cytoplasmic signalingsequences include, but are not limited to, those derived from CD3 zeta,common FcR gamma (FCER1G), Fc gamma RIIa, FcR beta (Fc Epsilon R1 b),CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, and DAP12.

The term “zeta” or alternatively “zeta chain”, “CD3-zeta” or “TCR-zeta”is defined as the protein provided as GenBan Acc. No. BAG36664.1, or theequivalent residues from a non-human species, e.g., mouse, rodent,monkey, ape and the like, and a “zeta stimulatory domain” oralternatively a “CD3-zeta stimulatory domain” or a “TCR-zeta stimulatorydomain” is defined as the amino acid residues from the cytoplasmicdomain of the zeta chain, or functional derivatives thereof, that aresufficient to functionally transmit an initial signal necessary for Tcell activation. In one aspect the cytoplasmic domain of zeta comprisesresidues 52 through 164 of GenBank Acc. No. BAG36664.1 or the equivalentresidues from a non-human species, e.g., mouse, rodent, monkey, ape andthe like, that are functional orthologs thereof.

The term a “costimulatory molecule” refers to a cognate binding partneron a T cell that specifically binds with a costimulatory ligand, therebymediating a costimulatory response by the T cell, such as, but notlimited to, proliferation. Costimulatory molecules are cell surfacemolecules other than antigen receptors or their ligands that contributeto an efficient immune response. Costimulatory molecules include, butare not limited to an MHC class I molecule, BTLA and a Toll ligandreceptor, as well as OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18),ICOS (CD278), and 4-1 BB (CD137). Further examples of such costimulatorymolecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80(KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8alpha, CD8beta, IL2Rbeta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D,ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1,ITGAM, CD11 b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7,NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4),CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1,CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150,IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76,PAG/Cbp, CD19a, and a ligand that specifically binds with CD83.

A costimulatory intracellular signaling domain can be the intracellularportion of a costimulatory molecule. A costimulatory molecule can berepresented in the following protein families: TNF receptor proteins,Immunoglobulin-like proteins, cytokine receptors, integrins, signalinglymphocytic activation molecules (SLAM proteins), and activating NK cellreceptors. Examples of such molecules include CD27, CD28, 4-1 BB(CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, ICAM-1, lymphocytefunction-associated antigen-1 (LFA-1), CD2, CDS, CD7, CD287, LIGHT,NKG2C, NKG2D, SLAMF7, NKp80, NKp30, NKp44, NKp46, CD160, B7-H3, and aligand that specifically binds with CD83, and the like.

The intracellular signaling domain can comprise the entire intracellularportion, or the entire native intracellular signaling domain, of themolecule from which it is derived, or a functional fragment orderivative thereof.

The term “4-1 BB” refers to a member of the TNFR superfamily with anamino acid sequence provided as GenBank Acc. No. AAA62478.2, or theequivalent residues from a non-human species, e.g., mouse, rodent,monkey, ape and the like; and a “4-1 BB costimulatory domain” is definedas amino acid residues 214-255 of GenBank Acc. No. AAA62478.2, or theequivalent residues from a non-human species, e.g., mouse, rodent,monkey, ape and the like.

The term “encoding” refers to the inherent property of specificsequences of nucleotides in a polynucleotide, such as a gene, a cDNA, oran mRNA, to serve as templates for synthesis of other polymers andmacromolecules in biological processes having either a defined sequenceof nucleotides (e.g., rRNA, tRNA and mRNA) or a defined sequence ofamino acids and the biological properties resulting therefrom. Thus, agene, cDNA, or RNA, encodes a protein if transcription and translationof mRNA corresponding to that gene produces the protein in a cell orother biological system. Both the coding strand, the nucleotide sequenceof which is identical to the mRNA sequence and is usually provided insequence listings, and the non-coding strand, used as the template fortranscription of a gene or cDNA, can be referred to as encoding theprotein or other product of that gene or cDNA.

The term “expression” refers to the transcription and/or translation ofa particular nucleotide sequence driven by a promoter.

The term “transfer vector” refers to a composition of matter whichcomprises an isolated nucleic acid and which can be used to deliver theisolated nucleic acid to the interior of a cell. Numerous vectors areknown in the art including, but not limited to, linear polynucleotides,polynucleotides associated with ionic or amphiphilic compounds,plasmids, and viruses. Thus, the term “transfer vector” includes anautonomously replicating plasmid. The term should also be construed tofurther include non-plasmid and non-viral compounds which facilitatetransfer of transgene into cells. In some instances, a transfer vectoris a plasmid DNA construct encoding transgene and lentiviralcis-elements required for packaging and insertion into host genome.

The term “lentivirus” refers to a genus of the Retroviridae family.Lentiviruses are unique among the retroviruses in being able to infectnon-dividing cells; they can deliver a significant amount of geneticinformation into the DNA of the host cell, so they are one of the mostefficient methods of a gene delivery vector. HIV, SIV, and FIV are allexamples of lentiviruses.

The term “lentiviral vector” refers to a vector including one or morenucleic acid sequences derived from at least a portion of a lentivirusgenome. A lentiviral vector may contains non-coding sequences of one ormore proteins from a lentivirus (e.g., HIV-1). A “lentiviral transfervector” may include a heterologous nucleic acid sequence, for example,to be transferred into a cell, and may further include, for example, oneor more lentiviral genes, or portions thereof. A “lentiviral packagingvector” may include one or more genes encoding lentiviral proteins, orportions thereof. For example, a lentiviral envelope protein may includea gene encoding an env protein, or a portion thereof. Transfection ofhost cells with a transfer vector and one or more packaging vectors canbe carried out in order to produce a virus, which in turn to be used toinfect target cells to express one or more transgenes comprised within aheterologous nucleic acid sequence within the target cells. A“transgene” thus refers to a heterologous gene that is transferred intoa target cell, for example, using a lentiviral transfer vector of theinvention. In certain examples described herein, a transgene is a geneencoding a chimeric antigen receptor, such as those described herein.

The term “isolated” means altered or removed from the natural state. Forexample, a nucleic acid or a peptide naturally present in a livinganimal is not “isolated,” but the same nucleic acid or peptide partiallyor completely separated from the coexisting materials of its naturalstate is “isolated.” An isolated nucleic acid or protein can exist insubstantially purified form, or can exist in a non-native environmentsuch as, for example, a host cell.

The term “nucleic acid” or “polynucleotide” refers to deoxyribonucleicacids (DNA) or ribonucleic acids (RNA) and polymers thereof in eithersingle- or double-stranded form. Unless specifically limited, the termencompasses nucleic acids containing known analogues of naturalnucleotides that have similar binding properties as the referencenucleic acid and are metabolized in a manner similar to naturallyoccurring nucleotides. Unless otherwise indicated, a particular nucleicacid sequence also implicitly encompasses conservatively modifiedvariants thereof (e.g., degenerate codon substitutions), alleles,orthologs, SNPs, and complementary sequences as well as the sequenceexplicitly indicated. Specifically, degenerate codon substitutions maybe achieved by generating sequences in which the third position of oneor more selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991);Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini etal., Mol. Cell. Probes 8:91-98 (1994)).

The terms “peptide,” “polypeptide,” and “protein” are usedinterchangeably, and refer to a compound comprised of amino acidresidues covalently linked by peptide bonds. A protein or peptide mustcontain at least two amino acids, and no limitation is placed on themaximum number of amino acids that can comprise a protein's or peptide'ssequence. Polypeptides include any peptide or protein comprising two ormore amino acids joined to each other by peptide bonds. As used herein,the term refers to both short chains, which also commonly are referredto in the art as peptides, oligopeptides and oligomers, for example, andto longer chains, which generally are referred to in the art asproteins, of which there are many types. “Polypeptides” include, forexample, biologically active fragments, substantially homologouspolypeptides, oligopeptides, homodimers, heterodimers, variants ofpolypeptides, modified polypeptides, derivatives, analogs, fusionproteins, among others. A polypeptide includes a natural peptide, arecombinant peptide, or a combination thereof.

The term “promoter” refers to a DNA sequence recognized by the syntheticmachinery of the cell, or introduced synthetic machinery, required toinitiate the specific transcription of a polynucleotide sequence.

The term “promoter/regulatory sequence” refers to a nucleic acidsequence which is required for expression of a gene product operablylinked to the promoter/regulatory sequence. In some instances, thissequence may be the core promoter sequence and in other instances, thissequence may also include an enhancer sequence and other regulatoryelements which are required for expression of the gene product. Thepromoter/regulatory sequence may, for example, be one which expressesthe gene product in a tissue specific manner.

As used herein, a “poly(A)” is a series of adenosines attached bypolyadenylation to the mRNA. In the preferred embodiment of a constructfor transient expression, the polyA is between 50 and 5000 nucleotidesin length, preferably greater than 64 nucleotides, more preferablygreater than 100 nucleotides, most preferably greater than 300 or 400nucleotides. Poly(A) sequences can be modified chemically orenzymatically to modulate mRNA functionality such as localization,stability or efficiency of translation.

As used herein, “polyadenylation” refers to the covalent linkage of apolyadenylyl moiety, or its modified variant, to a messenger RNAmolecule. In eukaryotic organisms, most messenger RNA (mRNA) moleculesare polyadenylated at the 3′ end. The 3′ poly(A) tail is a long sequenceof adenine nucleotides (often several hundred) added to the pre-mRNAthrough the action of an enzyme, polyadenylate polymerase. In highereukaryotes, the poly(A) tail is added onto transcripts that contain aspecific sequence, the polyadenylation signal. The poly(A) tail and theprotein bound to it aid in protecting mRNA from degradation byexonucleases. Polyadenylation is also important for transcriptiontermination, export of the mRNA from the nucleus, and translation.Polyadenylation occurs in the nucleus immediately after transcription ofDNA into RNA, but additionally can also occur later in the cytoplasm.After transcription has been terminated, the mRNA chain is cleavedthrough the action of an endonuclease complex associated with RNApolymerase. The cleavage site is usually characterized by the presenceof the base sequence AAUAAA near the cleavage site. After the mRNA hasbeen cleaved, adenosine residues are added to the free 3′ end at thecleavage site.

The term “subject” is intended to include a living organism (e.g.,mammals, human). In some instances, a subject may be a subject in whichan immune response can be elicited.

The term “transfected” or “transformed” or “transduced” refers to aprocess by which exogenous nucleic acid is transferred or introducedinto the host cell. A “transfected” or “transformed” or “transduced”cell is one which has been transfected, transformed, or transduced withexogenous nucleic acid. The cell includes the primary subject cell andits progeny. An “infected” cell is one that has be transfected,transformed, or transduced with a pathogen, for example, a virus (e.g.,a lentivirus). In some instances, one or more viral nucleic acidelements may be integrated into the genome of an infected cell.

The invention provides several advantages. For example, the viraltransfer vectors of the invention can be used to achieve increasedproduction of viral genomic RNA in packaging cells, increased nuclearexport of RNA, and increased viral titer. In addition, the viraltransfer vectors can be of a size that is relatively small, compared toprior vectors, which facilitates ease of use and permits the insertionof larger heterologous sequences.

Other features and advantages of the invention will be apparent from thefollowing detailed description, the drawings, and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic showing the feature map of the pCINS lentiviraltransfer vector. P-CMV=cytomegalovirus (CMV) promoter; R=LTR R region,U5=LTR U5 region, PBS=primer binding site; SD=major splice donor site;psi=packaging signal; RRE=Rev-response element; SA7=splice acceptorsite; cPPT=central polypurine tract; SA1=splice acceptor site;EF1A=human EF1alpha promoter; EGFP=GFP reporter open reading frame(ORF); WPRE=woodchuck hepatitis virus post-transcriptional regulatoryelement; SV40 polyA=SV40 polyadenylation sequence; nptII=kanamycinresistance gene; pUC ori=origin of replication from pUC.

FIG. 2 is a schematic showing the restriction map of the pCINSlentiviral transfer vector, including the location of each restrictionsite within the vector sequence.

FIG. 3 is a schematic showing the feature map of the pNOX lentiviraltransfer vector. P-CMV=cytomegalovirus (CMV) promoter; R=LTR R region,U5=LTR U5 region, PBS=primer binding site; SD=major splice donor site;psi=packaging signal; RRE=Rev-response element; SA7=splice acceptorsite; cPPT=central polypurine tract; SA1=splice acceptor site;EF1A=human EF1alpha promoter; EGFP=GFP reporter open reading frame(ORF); HPRE-NoX=hepatitis B post-transcriptional regulatory element,without ORF encoding X protein; dU3=truncated LTR U3 region; SV40polyA=SV40 polyadenylation sequence; nptII=kanamycin resistance gene;pUC ori=origin of replication from pUC.

FIG. 4 is a schematic showing the restriction map of the pNOX lentiviraltransfer vector, including the location of each restriction site withinthe vector sequence.

FIG. 5A-5E is a series of graphs showing the performance of theGFP-encoding pCINS and pNOX vectors relative to each other and incomparison to other lentiviral vectors. The following packaging vectorswere used in these experiments: pNVS-MDG-VSVG-Kan; pNVS-MDLgp-RRE; pNVSRSV Rev-Kan. FIG. 5A Use of the pCINS transfer vector results in viraltiter several times higher than that generated using the parentaltransfer vector before optimization. FIG. 5B The pCINS and pNOX vectorsgenerate similar viral titers in 293T cells. FIG. 5C pNOX generatedhigher viral titers in Jurkat cells compared to pCINS. FIG. 5DTransduction of primary human T cells with pCINS or pNOX resulted in thegeneration similar percentages of GFP-expressing T cells, indicatingthat pCINS and pNOX generate similar infectious titers in primary humanT cells. FIG. 5E A population of T cells that had been transduced withpNOX, which contains the HPRE post-transcriptional regulatory element,showed similar quantities of GFP+ cells after integration of lentiviralvector sequences compared to a population of T cells transduced withpCINS, which contains the WPRE post-transcriptional regulatory element.

FIG. 6 is a schematic showing certain features that were modified in astrategy undertaken to optimize a lentiviral production system.

FIG. 7A-7B is a set of graphs showing the performance of the parentaltransfer vector and pNLV transfer vectors relative to each other. FIG.7A Use of the pNLV lentiviral backbone produces more packaged lentiviralgenomic RNA than the control GFP vector lentiviral backbone, as measuredby qRT-PCR using Lenti-X qRT-PCR Titration Kit (Clontech). FIG. 7BTransduction of 293T cells with the pNLV vector generated higher viraltiters compared to the control transfer vector, as measured by proviralintegration assay using qPCR.

FIG. 8A-8C is a series of plots showing FACS measurements of transgeneexpression in SupT1 cells transduced with pCINS or pNLV lentiviralvectors expressing a transgene (e.g., a CAR). FIG. 8A FACS plot of cellstransduced with a pCINS lentiviral vector containing a transgene, usinga PE-conjugated antibody as a detection reagent for the transgene on thesurface. FIG. 8B FACS plot of cells transduced with a pNLV lentiviralvector containing a transgene, using a PE-conjugated antibody as adetection reagent for the transgene. FIG. 8C Plot comparing the MFImeasured in the FACS analyses of FIG. 8A and FIG. 8B, showing highersurface expression of the transgene in cells transduced with the pNLVtransfer vector.

FIG. 9A-9E is a series of graphs comparing the viral titers ofcommercial transfer vectors to a control vector, which is GFP-encodingSIN transfer vector before optimization, and to the pCINS and pNLVtransfer vectors. FIG. 9A The second generation pLVX (Clontech)lentiviral vector was produced with Tat-expressing construct andtransduced into 293T cells and was found to have a slightly higher viraltiter than a control transfer vector. FIG. 9B The pLenti6.2 (LifeTechnologies) lentiviral vector was transduced into cells and was foundto have a large decrease in viral titer compared to the control transfervector. FIG. 9C The pD2109 (DNA2.0) lentiviral vector was transducedinto cells and was found to have a small decrease in viral titercompared to the control transfer vector. FIG. 9D The pCINS lentiviralvector was transduced into cells and was found to have a much higherviral titer than the control transfer vector. FIG. 9E The pNLVlentiviral vector was transduced into cells and was found to have ahigher viral titer than the pCINS transfer vector.

FIG. 10A-10B is a schematic and plot comparing the effects of differentsplice site mutations in control non-optimized transfer construct onviral titer. FIG. 10A Panel A shows a schematic showing the lentiviralbackbone with labeled arrowheads denoting the location of the splicedonor and splice acceptor sites that were mutated for subsequent viraltiter determination. FIG. 10B The various splice donor and spliceacceptor site mutants of the indicated transfer vector were transducedinto cells and the viral titer was compared across the panel of mutantsto a control transfer vector. All of the mutations led to a largedecrease in viral titer, with the SA7mut and E-SAmut transfer vectorshaving slightly improved titers.

FIG. 11A-11B is a schematic and plot of a transfer vector backbonecontaining gag and/or env-derived sequence deletions and comparing thesubsequent effects on viral titer. FIG. 11A Panel A shows a schematic ofthe lentiviral backbone with annotations indicating the approximatelocation of gag and/or env deletions. FIG. 11B Transfer vectorconstructs with deletions in the env and/or gag regions were transducedinto cells and the viral titer was determined. Compared to a controltransfer vector, the -env3′ and -gag3′-env5′ mutants had a decrease inviral titer, while the -envy5′ and -gag3′ showed little difference inviral titer.

FIG. 12A-12F is a series of schematics representing several sequencealignments between the pNLV transfer vector and the pRRLSIN transfervector across key viral cis elements. FIG. 12A Panel A shows a schematicof the alignment between the psi regions of the two vectors, with theshaded regions denoting areas of sequence variation. FIG. 12B Panel Bshows a schematic of the alignment between the gag regions of the twovectors, with the shaded regions denoting areas of sequence variation.FIG. 12C Panel C shows a schematic of the alignment between the envregions of the two vectors, with the shaded regions denoting areas ofsequence variation. FIG. 12D Panel D shows a schematic of the alignmentbetween the RRE regions of the two vectors, with the shaded regionsdenoting areas of sequence variation. FIG. 12E Panel E shows a schematicof the alignment between the env (with major splice acceptor site 7)regions of the two vectors, with the shaded regions denoting areas ofsequence variation. FIG. 12F Panel F shows a schematic of the alignmentbetween the pol (with cPPT and major splice acceptor site 1) regions ofthe two vectors, with the shaded regions denoting areas of sequencevariation. #=Single-nucleotide substitution. Top row=pNLV; bottomrow=pRRLSIN. pRRLSIN sequence corresponds to the sequence of thepRRLSIN.cPPT.PGK-GFP.WPRE vector (Addgene Plasmid #12252). The pNLV cPPTsequence is that of SEQ ID NO:92.

FIG. 13 is a schematic showing a feature map of the pNLV lentiviraltransfer vector.

FIG. 14 is a schematic showing the restriction map of the pNLVlentiviral transfer vector, including the location of each restrictionsite within the vector sequence.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides lentiviral transfer vectors and usesthereof. Generally, the lentiviral transfer vectors of the inventioninclude a heterologous nucleic acid sequence, such as, for example, asequence encoding a transgene (e.g., a gene encoding a chimeric antigenreceptor (CAR); see below). The lentiviral transfer vectors furtherinclude a combination of two or more additional desirable features, asdescribed below.

The lentiviral transfer vectors of the invention can be useful, forexample, in providing a heterologous nucleic acid sequence to a hostcell for packaging into a virus, which in turn can be used forexpressing a transgene within the heterologous nucleic acid sequence ina desired target cell. For example, the lentiviral transfer vectors maybe introduced into a host cell in combination with, for example, one ormore packaging vectors, such that the host cell produces a lentivirus.The lentivirus can then be used, for example, to infect a desired targetcell. In certain instances, lentiviral infection of the desired targetcell may result in integration of one or more nucleic acid sequencesfrom the lentiviral transfer vector (e.g., a heterologous nucleic acidencoding a transgene) into the genome of the desired target cell (e.g.,a T cell). Thus, the transduced cell may be capable of expressing one ormore genes (e.g., a CAR gene) present in the heterologous nucleic acidand/or the viral elements, and this capability may be passed on to theprogeny of the transduced cell.

Introduction of nucleic acid sequences from the lentiviral transfervector of the invention into a target cell, via a lentivirus generatedin a host cell, can enable expression, by the target cell, of elementsfrom the lentiviral transfer vector (e.g., the heterologous nucleic acidencoding the transgene).

Transfer Vector Elements

Lentiviral transfer vectors of the invention include elements suitablefor enabling transfer of heterologous nucleic acids present in thelentiviral transfer vector into a cell (e.g., a cell transfected withthe lentiviral transfer vector, and optionally one or more additionalvectors, such as packaging vectors). In particular, lentiviral transfervectors of the invention are generally characterized by at least two ofthe following features: (a) including a cytomegalovirus (CMV) promoter;(b) including a polynucleotide encoding at least a portion of a gagprotein that includes a mutated INS1 inhibitory sequence that reducesrestriction of nuclear export of RNA relative to wild-type INS1; (c) notincluding a polynucleotide encoding INS2, INS3, and INS4 inhibitorysequences of gag; and (d) not including an SV40 origin of replicationand/or an f1 origin of replication. In some instances, a lentiviraltransfer vector of the invention includes a cPPT element having a lengthof about 178 nucleotides (e.g., about 100, 110, 120, 125, 130, 140, 150,160, 170, 175, 178, 180, 190, 200, 225, or 250 nucleotides). In oneinstance, the cPPT element has a length of 178 nucleotides. In certaininstances, the vector includes a Kozak sequence positioned upstream(e.g., immediately upstream) of a heterologous nucleic acid or transgeneto be transferred into a host cell.

In some instances, the lentiviral transfer vectors may include one ormore of the following: a promoter (e.g., a CMV, RSV, or EF1a promoter)driving expression of one or more viral sequences, long terminal repeat(LTR) regions (e.g., an R region or an U5 region), a primer binding site(PBS), a packaging signal (psi) (e.g., a packaging signal including amajor splice donor site (SD)), a partial gag sequence (e.g., asdescribed herein), a partial env sequence, a Rev-response element (RRE),additional partial env sequence, optionally including a splice acceptorsite (e.g., an SA7 splice acceptor), a partial pol sequence including acentral polypurine tract (cPPT) (e.g., a cPPT comprising a spliceacceptor site, e.g., SA1), a subgenomic promoter (e.g., P-EF1a), aheterologous nucleic acid (e.g., a heterologous nucleic acid including agene encoding EGFP and/or a transgene of interest (e.g., a CAR gene)), apost-transcriptional regulatory element (e.g., a WPRE or HPRE,optionally including an X protein mutation), a polyA sequence (e.g., anSV40 polyA tail), a selectable marker (e.g., a kanamycin resistance gene(nptII), ampicilin resistance gene, or a chloramphenicol resistancegene), and an origin of replication (e.g., a pUC origin of replication,an SV40 origin of replication, or an f1 origin of replication). As notedabove, the lentiviral transfer vectors may include an EF1a promoter,which can be used to drive expression of the transgene. In particularinstances, the EF1a promoter includes a wild-type EF1a promoter sequencehaving the nucleic acid sequence of SEQ ID NO: 95. In some instances,cells transfected with a lentiviral transfer vector of the inventionand/or a packaging vector as described herein produce lentiviral RNAthat is primarily spliced. Lentiviral vectors of the invention may alsoinclude additional sequences (e.g., vector backbone sequences), such aswell known in the art. In certain instances, lentiviral vectors of theinvention may include or incorporate sequences from the vectorsdescribed herein (e.g., pNOX, pCINS, and/or pNLV). In particularinstances, lentiviral vectors of the invention may include orincorporate vector backbone sequences, or portions thereof, from vectorsdescribed herein (e.g., pNOX, pCINS, and/or pNLV). In one example, alentiviral vector of the invention incorporates a vector backbonesequence, or portion thereof, from pNLV.

The lentiviral transfer vector may also include elements suitable fordriving expression of the heterologous protein in a cell. In certaininstances, a Kozak sequence is positioned upstream of the heterologousprotein open reading frame. For example, the lentiviral transfer vectormay include a promoter (e.g., a CMV, RSV, or EF1a promoter) thatcontrols the expression of the heterologous nucleic acid. Otherpromoters suitable for use in the lentiviral transfer vector include,for example, constitutive promoters or tissue/cell type-specificpromoters. In some instances, the lentiviral transfer vector includes ameans of selectively marking a gene product (e.g., a polypeptide or RNA)encoded by at least a portion of the heterologous nucleic acid (e.g., agene product of interest). For example, the lentiviral transfer vectormay include a marker gene (e.g., a gene encoding a selectable marker,such as a fluorescent protein (e.g., GFP, YFP, RFP, dsRed, mCherry, orany derivative thereof)). The marker gene may be expressed independentlyof the gene product of interest. Alternatively, the marker gene may beco-expressed with the gene product of interest. For example, the markergene may be under the control of the same or different promoter as thegene product of interest. In another example, the marker gene may befused to the gene product of interest. The elements of the lentiviraltransfer vectors of the invention are, in general, in operableassociation with one another, to enable the transfer vectors toparticipate in the formation of a lentivirus in a transfected cell,together with one or more packaging vectors.

Viral Proteins

In some instances, a lentiviral transfer vector of the invention mayinclude one or more genes encoding viral proteins, or portions thereof.For example, the lentiviral transfer vector may include a polynucleotideencoding at least a portion of a gag protein (e.g., an HIV-1 gagprotein). In various examples, the sequence encoding the gag proteincomprises 250 nucleotides or less, e.g., 200 nucleotides or less, 175nucleotides or less, or 150 nucleotides or less. In one example, thesequence encoding the gag protein comprises or consists of 168nucleotides. The nucleotide sequence encoding gag can include INS1sequences (e.g., with the mutations noted below), but lack INS2, INS3,and INS4 sequences. The polynucleotide encoding the gag protein, orportion thereof, may include mutations that inactivate one or moreinhibitory sequences, for example, as described herein. Mutations in oneor more (e.g., all) of the following nucleotides of the INS1 region canbe included: G989, G992, C995, G998, C999, G1004, C1007T, and C1010(using the pNLV sequence of FIG. 12B as a reference (pNLV)). In specificexamples, these mutations are as follows: G989A, G992A, C995T, G998A,C999T, G1004A, C1007T, and C1010A. Furthermore, sequences encoding gagcan include an insertion resulting in a frameshift and prematuretermination (see, e.g., FIG. 12B, below) of undesirable production ofgag protein. In certain instances, a lentiviral transfer vector mayinclude a partial gag sequence (e.g., a partial gag sequence positionedadjacent to and/or overlapping with a packaging signal in the lentiviraltransfer vector), a partial env sequence, and/or a partial pol sequence(e.g., a central polypurine tract from pol). The partial gag, partialenv, and/or partial pol sequences may, in certain instances, originatefrom HIV-1 (e.g., HIV-1 isolate NL4-3 or SF3). In one example, thelentiviral transfer vector may include a partial gag sequence under thecontrol of a CMV promoter.

Post-Transcriptional Regulatory Elements (PREs)

A lentiviral transfer vector of the invention may include apost-transcriptional regulatory element (PRE). PREs are nucleic acidsequences that contribute to regulation of expression of a DNA sequencewithin which the PRE is located. For example, a PRE may be transcribedalong with the rest of the DNA sequence. The portion of the resultingmRNA molecule transcribed from the PRE may form a tertiary structurethat enhances expression of the gene product. A PRE may include, in someinstances, three components (alpha, beta, and gamma). The activity ofthe PRE may depend on how many of the components are present. Forexample, a full tripartite PRE may be more active than the alphacomponent alone. PREs suitable for inclusion in the lentiviral transfervectors of the invention include, for example, woodchuck hepatitis virusPRE (WPRE) and/or hepatitis B virus PRE (HPRE). In certain instances, anHPRE may include a natural HPRE sequence (e.g., a natural HPRE sequencederived from hepatitis B virus isolate bba6, complete genome; GenBank:KP341007.1). In some instances, a PRE in a lentiviral transfer vector ofthe invention may be modified to inactivate an X protein, as describedherein.

Omitted Elements

The present invention features lentiviral transfer vectors that havebeen optimized over existing vectors. In some instances, it is desirableto reduce the size of a vector, such as a lentiviral transfer vector ofthe invention. For example, redundant sequences or elements may beremoved from a vector to reduce its size. In certain instances, unneededorigins of replication (e.g., an SV40 ori sequence and/or an f1 orisequence) may be removed from a lentiviral vector. Thus, in variousexamples, the lentiviral transfer vectors of the invention may be lessthan 8000 nucleotides in length, e.g., less than 7900, 7800, or 7700nucleotides in length.

In some instances, portions of elements or genes encoded by a vector maybe deleted from the vector. For example, protein-coding genes mayinclude inhibitory sequences. Such inhibitory sequences may inhibit theexpression, processing, and/or function of a gene. For example, theHIV-1 gag protein includes a series of inhibitory RNA elements known asINS elements (e.g., INS1, INS2, INS3, and INS4). Such INS elements aredescribed, for example, in J. Virol. 71(7):4892-4903, 1997; J. Virol.66(12):7176-7182, 1992; and J. Virol. 68(6):3784-3793, 1994; each ofwhich is incorporated herein by reference. In one example, INS1 isinvolved in restricting nuclear export of unspliced viral RNA (see,e.g., J. Virol. 66(12):7176-7182, 1992). Removal or mutation of one ormore (e.g., 1, 2, 3, or 4) of these inhibitory sequences (e.g., bymutation of the nucleotides forming a particular INS element; see, e.g.,above) from a gene (e.g., a gene encoding a gag protein, or a portionthereof) may therefore increase the expression, processing, and/orfunction of the gene, or its gene product. In one example, mutation ofthe INS1 element in a gene encoding a gag protein, or a portion thereof,results in increased nuclear export of unspliced viral RNAs.

In some instances, entire elements or genes encoded by a vector may bedeleted from the vector. For example, some post-transcriptionalregulatory elements, such as WPRE, include a polynucleotide encoding anX protein, or a portion thereof. The X protein has been implicated ingeneration of liver cancers (see, e.g., Gene Ther. 12(1):3-4, 2005;incorporated herein by reference). As such, it may be beneficial from abiosafety standpoint to prevent X protein activity, function, and/orexpression from the PRE of a lentiviral transfer vector of theinvention. For example, the X protein-encoding gene may be deleted fromthe vector. Alternatively, the start codon of the X protein-encodinggene may be mutated (e.g., from ATG to AGG), thereby preventingtranslation of the X protein. In another alternative, one or moreinactivating mutations may be introduced into the X protein amino acidsequence that prevents its function. Further approaches for inactivatingthe X protein include mutation methods well known in the art.

Packaging Vectors

The lentiviral transfer vectors of the invention may be co-transfectedinto a cell together with one or more additional vectors. In someinstances, the one or more additional vectors may include lentiviralpackaging vectors. In certain instances, the one or more additionalplasmids may include an envelope plasmid (e.g., an envelope plasmidencoding VSV-G, such as pMD.G). Generally, a packaging vector includesone or more polynucleotide sequences encoding lentiviral proteins (e.g.,gag, pol, env, tat, rev, vif, vpu, vpr, and/or nef protein, or aderivative, combination, or portion thereof). A packaging vector to beco-transfected into a cell with a lentiviral transfer vector of theinvention may include sequence(s) encoding one or more lentiviralproteins not encoded by the lentiviral transfer vector. For example, alentiviral transfer vector may be co-transfected with a first packagingvector encoding gag and pol and a second packaging vector encoding rev.Thus, co-transfection of a lentiviral transfer vector with suchpackaging vector(s) may result in the introduction of all genes requiredfor viral particle formation into the cell, thereby enabling the cell toproduce viral particles that may be isolated. Appropriate packagingvectors for use in the invention can be selected by those of skill inthe art based on, for example, consideration of the features selectedfor a lentiviral transfer vector of the invention. For examples ofpackaging vectors that can be used or adapted for use in the inventionsee, e.g., WO 03/064665, WO 2009/153563, U.S. Pat. No. 7,419,829, WO2004/022761, U.S. Pat. No. 5,817,491, WO 99/41397, U.S. Pat. Nos.6,924,123, 7,056,699, WO 99/32646, WO 98/51810, and WO 98/17815. In someinstances, a packaging plasmid may encode a gag and/or pol protein, andmay optionally include an RRE sequence (e.g., an pMDLgpRRE vector; see,e.g., Dull et al., J. Virol. 72(11):8463-8471, 1998). In certaininstances, a packaging vector may encode a rev protein (e.g., a pRSV-Revvector).

Host Cells for Lentivirus Production

A lentiviral transfer vector of the invention may be introduced into ahost cell (packaging cell). The lentiviral transfer vector is generallyco-transfected into the host cell together with one or more additionalvectors (e.g., one or more packaging vectors). The one or moreadditional vectors may encode viral proteins and/or regulatory proteins.Co-transfection of the lentiviral transfer vector and the one or moreadditional vectors enables the host cell to produce a lentivirus (e.g.,a lentivirus encoding a heterologous nucleic acid sequence from thelentiviral transfer vector). Lentiviruses produced by a host cell asdescribed herein may be used to infect another cell. The heterologousnucleic acid and/or one or more additional elements (e.g., promoters andviral elements) may be integrated into the genome of the infected cell,thereby permitting the cell and its progeny to express gene(s)originating from the lentiviral transfer vector.

A packaging cell suitable for transfection with the lentiviral transfervector (and one or more packaging vectors) may be a eukaryotic cell,such as a mammalian cell. The host cell may originate from a cell line(e.g., an immortalized cell line). For example, the host cell may be aHEK 293 cell (e.g., a 293T cell including the SV40 large T-antigen).Information regarding cells that are transduced by the lentiviruses isprovided below.

Target cell is the cell, which is infected (transduced) with lentiviralvector (lentivirus) encoding transgene of interest. After transduction,transgene of interest is stably inserted into target cell genome and canbe detected by molecular biology methods such as PCR and Southern blot.Transgene can be expressed in target cell and detected by flow cytometryor Western blot. In some instances, target cell is a human cell. Incertain instances, the host cell is a particular cell type of interest,e.g., a primary T cell, SupT1 cell, Jurkat cell, or 293T cell.

Methods of Producing Lentiviruses

The lentiviral transfer vectors of the invention may be useful forproducing lentiviruses in cells (e.g., a host cell as described herein).A method of producing a lentivirus using a lentiviral transfer vectordescribed herein will generally involve introducing the lentiviraltransfer vector and one or more additional vectors (e.g., a lentiviralpackaging vector) into the cell. The vectors may be introduced into thecell using transfection methods well known in the art. Aftertransfection, the cell may be permitted to express viral proteinsencoded by the lentiviral transfer vector and/or the one or moreadditional vectors (e.g., by incubating the cell under standardconditions known in the art for inducing viral gene expression). In someinstances, the viral genes are expressed under the control of aconstitutive or inducible promoter. In the latter case, viral geneexpression may be selectively induced by incubating the cell underconditions suitable for activating the inducible promoter. Viralproteins produced by the cell may subsequently form a viral particle,which buds from the cell surface and can be isolated from the solution(e.g., according to methods well known in the art). During formation ofthe virus, a polynucleotide including the sequence of the heterologousnucleic acid may be incorporated into the viral particle. Thus, thisprocess yields a lentivirus that includes the heterologous nucleic acidoriginating from the lentiviral transfer vector.

Heterologous Nucleic Acids

The present invention features a lentiviral transfer vector thatincludes a heterologous nucleic acid. The heterologous nucleic acid mayinclude a transgene of interest (e.g., a gene encoding a polypeptide ora gene for a noncoding RNA) that is to be expressed in a cell. In someinstances, the heterologous protein ORF is positioned downstream of aKozak sequence. The gene of interest may be, in certain instances,associated with (e.g., fused to) a marker gene, as described herein. Insome instances, the heterologous nucleic acid of the lentiviral transfervector will be present in a lentivirus produced in a cell transfectedwith the lentiviral transfer vector and, optionally, one or moreadditional vectors (e.g., packaging vectors). In certain instances, theheterologous nucleic acid may be integrated into the genome of a cellinfected with the lentivirus. Integration of the heterologous nucleicacid into the genome of such a cell may permit the cell and its progenyto express the gene of interest. The gene of interest may be any geneknown in the art. Exemplary genes of interest include, withoutlimitation, genes encoding chimeric antigen receptors (CARs), bindingmoieties (e.g., antibodies and antibody fragments), signaling proteins,cell surface proteins (e.g., T cell receptors), proteins involved indisease (e.g., cancers, autoimmune diseases, neurological disorders, orany other disease known in the art), or any derivative or combinationthereof.

Chimeric Antigen Receptors

A lentiviral transfer vector of the invention may be used to induce theproduction of a chimeric antigen receptor (CAR) in a cell. A CAR may bea transmembrane protein including (i) an extracellular antigen bindingdomain, (ii) a transmembrane domain, and (iii) an intracellularsignaling domain. A CAR encoded by a lentiviral transfer vector of thepresent invention may be any CAR known in the art. In one embodiment,the CAR includes an anti-CD19 antibody or a fragment thereof, a 4-1 BB(CD137) transmembrane domain, and a CD3-zeta signaling domain.

Antigen Binding Domain

The present invention can be used to make immune effector cells (e.g., Tcells, NK cells) that are engineered to contain one or more CARs thatdirect the immune effector cells to undesired cells (e.g., cancercells). This is achieved through an antigen binding domain on the CARthat is specific for a cancer associated antigen. There are two classesof cancer associated antigens (tumor antigens) that can be targeted bythe CARs of the instant invention: (1) cancer associated antigens thatare expressed on the surface of cancer cells; and (2) cancer associatedantigens that itself is intracelluar, however, a fragment of suchantigen (peptide) is presented on the surface of the cancer cells by MHC(major histocompatibility complex).

In some instances, the antigen binding domain is capable of specificallybinding to an antigen selected from the group consisting of CD19; CD123;CD22; CD30; CD171; CS-1; C-type lectin-like molecule-1, CD33; epidermalgrowth factor receptor variant III (EGFRvIII); ganglioside G2 (GD2);ganglioside GD3; TNF receptor family member B cell maturation (BCMA); Tnantigen ((Tn Ag) or (GaINAcα-Ser/Thr)); prostate-specific membraneantigen (PSMA); Receptor tyrosine kinase-like orphan receptor 1 (ROR1);Fms-Like Tyrosine Kinase 3 (FLT3); Tumor-associated glycoprotein 72(TAG72); CD38; CD44v6; Carcinoembryonic antigen (CEA); Epithelial celladhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); Interleukin-13receptor subunit alpha-2; mesothelin; Interleukin 11 receptor alpha(IL-11 Ra); prostate stem cell antigen (PSCA); Protease Serine 21;vascular endothelial growth factor receptor 2 (VEGFR2); Lewis(Y)antigen; CD24; Platelet-derived growth factor receptor beta(PDGFR-beta); Stage-specific embryonic antigen-4 (SSEA-4); CD20; Folatereceptor alpha; Receptor tyrosine-protein kinase ERBB2 (Her2/neu); Mucin1, cell surface associated (MUC1); epidermal growth factor receptor(EGFR); neural cell adhesion molecule (NCAM); Prostase; prostatic acidphosphatase (PAP); elongation factor 2 mutated (ELF2M); Ephrin B2;fibroblast activation protein alpha (FAP); insulin-like growth factor 1receptor (IGF-I receptor), carbonic anhydrase IX (CAIX); Proteasome(Prosome, Macropain) Subunit, Beta Type, 9 (LMP2); glycoprotein 100(gp100); oncogene fusion protein consisting of breakpoint cluster region(BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl)(bcr-abl); tyrosinase; ephrin type-A receptor 2 (EphA2); Fucosyl GM1;sialyl Lewis adhesion molecule (sLe); ganglioside GM3; transglutaminase5 (TGS5); high molecular weight-melanoma-associated antigen (HMWMAA);o-acetyl-GD2 ganglioside (OAcGD2); Folate receptor beta; tumorendothelial marker 1 (TEM1/CD248); tumor endothelial marker 7-related(TEM7R); claudin 6 (CLDN6); thyroid stimulating hormone receptor (TSHR);G protein-coupled receptor class C group 5, member D (GPRC5D);chromosome X open reading frame 61 (CXORF61); CD97; CD179a; anaplasticlymphoma kinase (ALK); Polysialic acid; placenta-specific 1 (PLAC1);hexasaccharide portion of globoH glycoceramide (GloboH); mammary glanddifferentiation antigen (NY-BR-1); uroplakin 2 (UPK2); Hepatitis A viruscellular receptor 1 (HAVCR1); adrenoceptor beta 3 (ADRB3); pannexin 3(PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6complex, locus K 9 (LY6K); Olfactory receptor 51E2 (OR51E2); TCR GammaAlternate Reading Frame Protein (TARP); Wilms tumor protein (WT1);Cancer/testis antigen 1 (NY-ESO-1); Cancer/testis antigen 2 (LAGE-1a);Melanoma-associated antigen 1 (MAGE-A1); ETS translocation-variant gene6, located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); XAntigen Family, Member 1A (XAGE1); angiopoietin-binding cell surfacereceptor 2 (Tie 2); melanoma cancer testis antigen-1 (MAD-CT-1);melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1;tumor protein p53 (p53); p53 mutant; prostein; surviving; telomerase;prostate carcinoma tumor antigen-1, melanoma antigen recognized by Tcells 1; Rat sarcoma (Ras) mutant; human Telomerase reversetranscriptase (hTERT); sarcoma translocation breakpoints; melanomainhibitor of apoptosis (ML-IAP); ERG (transmembrane protease, serine 2(TMPRSS2) ETS fusion gene); N-Acetyl glucosaminyl-transferase V (NA17);paired box protein Pax-3 (PAX3); Androgen receptor; Cyclin B1; v-mycavian myelocytomatosis viral oncogene neuroblastoma derived homolog(MYCN); Ras Homolog Family Member C (RhoC); Tyrosinase-related protein 2(TRP-2); Cytochrome P450 1B1 (CYP1B1); CCCTC-Binding Factor (Zinc FingerProtein)-Like, Squamous Cell Carcinoma Antigen Recognized By T Cells 3(SART3); Paired box protein Pax-5 (PAX5); proacrosin binding proteinsp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK); Akinase anchor protein 4 (AKAP-4); synovial sarcoma, X breakpoint 2(SSX2); Receptor for Advanced Glycation Endproducts (RAGE-1); renalubiquitous 1 (RU1); renal ubiquitous 2 (RU2); legumain; human papillomavirus E6 (HPV E6); human papilloma virus E7 (HPV E7); intestinalcarboxyl esterase; heat shock protein 70-2 mutated (mut hsp70-2); CD79a;CD79b; CD72; Leukocyte-associated immunoglobulin-like receptor 1(LAIR1); Fc fragment of IgA receptor (FCAR or CD89); Leukocyteimmunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300molecule-like family member f (CD300LF); C-type lectin domain family 12member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-likemodule-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyteantigen 75 (LY75); Glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); andimmunoglobulin lambda-like polypeptide 1 (IGLL1).

A CAR described herein can comprise an antigen binding domain (e.g.,antibody or antibody fragment, TCR or TCR fragment) that binds to atumor-supporting antigen (e.g., a tumor-supporting antigen as describedherein). In some embodiments, the tumor-supporting antigen is an antigenpresent on a stromal cell or a myeloid-derived suppressor cell (MDSC).Stromal cells can secrete growth factors to promote cell division in themicroenvironment. MDSC cells can inhibit T cell proliferation andactivation. Without wishing to be bound by theory, in some embodiments,the CAR-expressing cells destroy the tumor-supporting cells, therebyindirectly inhibiting tumor growth or survival.

In embodiments, the stromal cell antigen is chosen from one or more of:bone marrow stromal cell antigen 2 (BST2), fibroblast activation protein(FAP) and tenascin. In an embodiment, the FAP-specific antibody is,competes for binding with, or has the same CDRs as, sibrotuzumab. Inembodiments, the MDSC antigen is chosen from one or more of: CD33,CD11b, C14, CD15, and CD66b. Accordingly, in some embodiments, thetumor-supporting antigen is chosen from one or more of: bone marrowstromal cell antigen 2 (BST2), fibroblast activation protein (FAP) ortenascin, CD33, CD11b, C14, CD15, and CD66b.

Antigen Binding Domain Structures

The antigen binding domain of a CAR may include, for example, anypolypeptide binding moiety known in the art. For example, the antigenbinding domain may include an antibody or antibody fragment (e.g., anscFv, a Fv, a Fab, a (Fab′)2, a single domain antibody (SDAB), a VH orVL domain, a camelid VHH domain or a bi-functional (e.g. bi-specific)hybrid antibody). In preferred instances, the antigen binding domainincludes an scFv. In some instances, the antigen binding domain mayinclude the antigen binding domain is a T cell receptor (TCR), or afragment thereof, for example, a single chain TCR (scTCR). In certaininstances, the antigen binding domain is a bi- or multi-specificmolecule (e.g., a multispecific antibody molecule). In some instances,the antigen binding domain recognizes one or more particular targetmolecule(s) of interest. A target molecule of interest may be, forexample, associated with a disease or the development thereof.

In some instances, scFvs can be prepared according to method known inthe art (see, for example, Bird et al., (1988) Science 242:423-426 andHuston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). ScFvmolecules can be produced by linking VH and VL regions together usingflexible polypeptide linkers. The scFv molecules comprise a linker(e.g., a Ser-Gly linker) with an optimized length and/or amino acidcomposition. The linker length can greatly affect how the variableregions of a scFv fold and interact. In fact, if a short polypeptidelinker is employed (e.g., between 5-10 amino acids) intrachain foldingis prevented. Interchain folding is also required to bring the twovariable regions together to form a functional epitope binding site. Forexamples of linker orientation and size see, e.g., Hollinger et al. 1993Proc Natl Acad. Sci. U.S.A. 90:6444-6448, U.S. Patent ApplicationPublication Nos. 2005/0100543, 2005/0175606, 2007/0014794, and PCTpublication Nos. WO2006/020258 and WO2007/024715, is incorporated hereinby reference.

An scFv can comprise a linker of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, or moreamino acid residues between its VL and VH regions. The linker sequencemay comprise any naturally occurring amino acid. In some embodiments,the linker sequence comprises amino acids glycine and serine. In anotherembodiment, the linker sequence comprises sets of glycine and serinerepeats such as (Gly₄Ser)n, where n is a positive integer equal to orgreater than 1 (SEQ ID NO: 22). In one embodiment, the linker can be(Gly₄Ser)₄ (SEQ ID NO: 29) or (Gly₄Ser)₃ (SEQ ID NO: 30). Variation inthe linker length may retain or enhance activity, giving rise tosuperior efficacy in activity studies.

In another aspect, the antigen binding domain is a T cell receptor(“TCR”), or a fragment thereof, for example, a single chain TCR (scTCR).Methods to make such TCRs are known in the art. See, e.g., Willemsen R Aet al, Gene Therapy 7: 1369-1377 (2000); Zhang T et al, Cancer Gene Ther11: 487-496 (2004); Aggen et al, Gene Ther. 19(4):365-74 (2012)(references are incorporated herein by its entirety). For example, scTCRcan be engineered that contains the Vα and Vβ genes from a T cell clonelinked by a linker (e.g., a flexible peptide). This approach is veryuseful to cancer associated target that itself is intracellar, however,a fragment of such antigen (peptide) is presented on the surface of thecancer cells by MHC.

In certain embodiments, the encoded antigen binding domain has a bindingaffinity KD of 10⁻⁴ M to 10⁻⁸ M.

In one embodiment, the encoded CAR molecule comprises an antigen bindingdomain that has a binding affinity KD of 10⁻⁴ M to 10⁻⁸ M, e.g., 10⁻⁵ Mto 10⁻⁷ M, e.g., 10⁻⁶ M or 10⁻⁷ M, for the target antigen. In oneembodiment, the antigen binding domain has a binding affinity that is atleast five-fold, 10-fold, 20-fold, 30-fold, 50-fold, 100-fold or1,000-fold less than a reference antibody, e.g., an antibody describedherein. In one embodiment, the encoded antigen binding domain has abinding affinity at least 5-fold less than a reference antibody (e.g.,an antibody from which the antigen binding domain is derived). In oneaspect such antibody fragments are functional in that they provide abiological response that can include, but is not limited to, activationof an immune response, inhibition of signal-transduction originationfrom its target antigen, inhibition of kinase activity, and the like, aswill be understood by a skilled artisan. In one aspect, the antigenbinding domain of the CAR is a scFv antibody fragment that is humanizedcompared to the murine sequence of the scFv from which it is derived.

In one aspect, the antigen binding domain of a CAR of the invention(e.g., a scFv) is encoded by a nucleic acid molecule whose sequence hasbeen codon optimized for expression in a mammalian cell.

In one aspect, entire CAR construct of the invention is encoded by anucleic acid molecule whose entire sequence has been codon optimized forexpression in a mammalian cell. Codon optimization refers to thediscovery that the frequency of occurrence of synonymous codons (i.e.,codons that code for the same amino acid) in coding DNA is biased indifferent species. Such codon degeneracy allows an identical polypeptideto be encoded by a variety of nucleotide sequences. A variety of codonoptimization methods is known in the art, and include, e.g., methodsdisclosed in at least U.S. Pat. Nos. 5,786,464 and 6,114,148.

Specific Antigen Antibody Pairs

In one embodiment, an antigen binding domain against CD22 is an antigenbinding portion, e.g., CDRs, of an antibody described in, e.g., Haso etal., Blood, 121(7): 1165-1174 (2013); Wayne et al., Clin Cancer Res16(6): 1894-1903 (2010); Kato et al., Leuk Res 37(1):83-88 (2013);Creative BioMart (creativebiomart.net): MOM-18047-S(P).

In one embodiment, an antigen binding domain against CS-1 is an antigenbinding portion, e.g., CDRs, of Elotuzumab (BMS), see e.g., Tai et al.,2008, Blood 112(4):1329-37; Tai et al., 2007, Blood. 110(5):1656-63.

In one embodiment, an antigen binding domain against CLL-1 is an antigenbinding portion, e.g., CDRs, of an antibody available from R&D,ebiosciences, Abcam, for example, PE-CLL1-hu Cat #353604 (BioLegend);and PE-CLL1 (CLEC12A) Cat #562566 (BD).

In one embodiment, an antigen binding domain against CD33 is an antigenbinding portion, e.g., CDRs, of an antibody described in, e.g., Bross etal., Clin Cancer Res 7(6):1490-1496 (2001) (Gemtuzumab Ozogamicin,hP67.6), Caron et al., Cancer Res 52(24):6761-6767 (1992) (Lintuzumab,HuM195), Lapusan et al., Invest New Drugs 30(3):1121-1131 (2012)(AVE9633), Aigner et al., Leukemia 27(5): 1107-1115 (2013) (AMG330, CD33BiTE), Dutour et al., Adv hematol 2012:683065 (2012), and Pizzitola etal., Leukemia doi:10.1038/Lue.2014.62 (2014).

In one embodiment, an antigen binding domain against GD2 is an antigenbinding portion, e.g., CDRs, of an antibody described in, e.g., Mujoo etal., Cancer Res. 47(4)1098-1104 (1987); Cheung et al., Cancer Res45(6):2642-2649 (1985), Cheung et al., J Clin Oncol 5(9):1430-1440(1987), Cheung et al., J Clin Oncol 16(9):3053-3060 (1998),Handgretinger et al., Cancer Immunol Immunother 35(3):199-204 (1992). Insome embodiments, an antigen binding domain against GD2 is an antigenbinding portion of an antibody selected from mAb 14.18, 14G2a, ch14.18,hu14.18, 3F8, hu3F8, 3G6, 8B6, 60C3, 10B8, ME36.1, and 8H9, see e.g.,WO2012033885, WO2013040371, WO2013192294, WO2013061273, WO2013123061,WO2013074916, and WO201385552. In some embodiments, an antigen bindingdomain against GD2 is an antigen binding portion of an antibodydescribed in US Publication No.: 20100150910 or PCT Publication No.: WO2011160119.

In one embodiment, an antigen binding domain against BCMA is an antigenbinding portion, e.g., CDRs, of an antibody described in, e.g.,WO2012163805, WO200112812, and WO2003062401.

In one embodiment, an antigen binding domain against Tn antigen is anantigen binding portion, e.g., CDRs, of an antibody described in, e.g.,U.S. Pat. No. 8,440,798, Brooks et al., PNAS 107(22):10056-10061 (2010),and Stone et al., OncoImmunology 1(6):863-873(2012).

In one embodiment, an antigen binding domain against PSMA is an antigenbinding portion, e.g., CDRs, of an antibody described in, e.g., Parkeret al., Protein Expr Purif 89(2):136-145 (2013), US 20110268656 (J591ScFv); Frigerio et al, European J Cancer 49(9):2223-2232 (2013)(scFvD2B); WO 2006125481 (mAbs 3/A12, 3/E7 and 3/F11) and single chainantibody fragments (scFv A5 and D7).

In one embodiment, an antigen binding domain against ROR1 is an antigenbinding portion, e.g., CDRs, of an antibody described in, e.g., Hudeceket al., Clin Cancer Res 19(12):3153-3164 (2013); WO 2011159847; andUS20130101607.

In one embodiment, an antigen binding domain against FLT3 is an antigenbinding portion, e.g., CDRs, of an antibody described in, e.g.,WO2011076922, U.S. Pat. No. 5,777,084, EP0754230, US20090297529, andseveral commercial catalog antibodies (R&D, ebiosciences, Abcam).

In one embodiment, an antigen binding domain against TAG72 is an antigenbinding portion, e.g., CDRs, of an antibody described in, e.g., Hombachet al., Gastroenterology 113(4):1163-1170 (1997); and Abcam ab691.

In one embodiment, an antigen binding domain against FAP is an antigenbinding portion, e.g., CDRs, of an antibody described in, e.g.,Ostermann et al., Clinical Cancer Research 14:4584-4592 (2008) (FAP5),US Pat. Publication No. 2009/0304718; sibrotuzumab (see e.g., Hofheinzet al., Oncology Research and Treatment 26(1), 2003); and Tran et al., JExp Med 210(6):1125-1135 (2013).

In one embodiment, an antigen binding domain against CD38 is an antigenbinding portion, e.g., CDRs, of daratumumab (see, e.g., Groen et al.,Blood 116(21):1261-1262 (2010); MOR202 (see, e.g., U.S. Pat. No.8,263,746); or antibodies described in U.S. Pat. No. 8,362,211.

In one embodiment, an antigen binding domain against CD44v6 is anantigen binding portion, e.g., CDRs, of an antibody described in, e.g.,Casucci et al., Blood 122(20):3461-3472 (2013).

In one embodiment, an antigen binding domain against CEA is an antigenbinding portion, e.g., CDRs, of an antibody described in, e.g.,Chmielewski et al., Gastoenterology 143(4):1095-1107 (2012).

In one embodiment, an antigen binding domain against EPCAM is an antigenbinding portion, e.g., CDRS, of an antibody selected from MT110,EpCAM-CD3 bispecific Ab (see, e.g.,clinicaltrials.gov/ct2/show/NCT00635596); Edrecolomab; 3622W94; ING-1;and adecatumumab (MT201).

In one embodiment, an antigen binding domain against PRSS21 is anantigen binding portion, e.g., CDRs, of an antibody described in U.S.Pat. No. 8,080,650.

In one embodiment, an antigen binding domain against B7H3 is an antigenbinding portion, e.g., CDRs, of an antibody MGA271 (Macrogenics).

In one embodiment, an antigen binding domain against KIT is an antigenbinding portion, e.g., CDRs, of an antibody described in, e.g., U.S.Pat. No. 7,915,391, US20120288506, and several commercial catalogantibodies.

In one embodiment, an antigen binding domain against IL-13Ra2 is anantigen binding portion, e.g., CDRs, of an antibody described in, e.g.,WO2008/146911, WO2004087758, several commercial catalog antibodies, andWO2004087758.

In one embodiment, an antigen binding domain against CD30 is an antigenbinding portion, e.g., CDRs, of an antibody described in, e.g., U.S.Pat. No. 7,090,843 B1, and EP0805871.

In one embodiment, an antigen binding domain against GD3 is an antigenbinding portion, e.g., CDRs, of an antibody described in, e.g., U.S.Pat. Nos. 7,253,263; 8,207,308; US 20120276046; EP1013761; WO2005035577;and U.S. Pat. No. 6,437,098.

In one embodiment, an antigen binding domain against CD171 is an antigenbinding portion, e.g., CDRs, of an antibody described in, e.g., Hong etal., J Immunother 37(2):93-104 (2014).

In one embodiment, an antigen binding domain against IL-11 Ra is anantigen binding portion, e.g., CDRs, of an antibody available from Abcam(cat #ab55262) or Novus Biologicals (cat #EPR5446). In anotherembodiment, an antigen binding domain again IL-11 Ra is a peptide, see,e.g., Huang et al., Cancer Res 72(1):271-281 (2012).

In one embodiment, an antigen binding domain against PSCA is an antigenbinding portion, e.g., CDRs, of an antibody described in, e.g.,Morgenroth et al., Prostate 67(10):1121-1131 (2007) (scFv 7F5);Nejatollahi et al., J of Oncology 2013(2013), article ID 839831 (scFvC5-II); and US Pat Publication No. 20090311181.

In one embodiment, an antigen binding domain against VEGFR2 is anantigen binding portion, e.g., CDRs, of an antibody described in, e.g.,Chinnasamy et al., J Clin Invest 120(11):3953-3968 (2010).

In one embodiment, an antigen binding domain against LewisY is anantigen binding portion, e.g., CDRs, of an antibody described in, e.g.,Kelly et al., Cancer Biother Radiopharm 23(4):411-423 (2008) (hu3S193 Ab(scFvs)); Dolezal et al., Protein Engineering 16(1):47-56 (2003) (NC10scFv).

In one embodiment, an antigen binding domain against CD24 is an antigenbinding portion, e.g., CDRs, of an antibody described in, e.g., Maliaret al., Gastroenterology 143(5):1375-1384 (2012).

In one embodiment, an antigen binding domain against PDGFR-beta is anantigen binding portion, e.g., CDRs, of an antibody Abcam ab32570.

In one embodiment, an antigen binding domain against SSEA-4 is anantigen binding portion, e.g., CDRs, of antibody MC813 (Cell Signaling),or other commercially available antibodies.

In one embodiment, an antigen binding domain against CD20 is an antigenbinding portion, e.g., CDRs, of the antibody Rituximab, Ofatumumab,Ocrelizumab, Veltuzumab, or GA101.

In one embodiment, an antigen binding domain against Folate receptoralpha is an antigen binding portion, e.g., CDRs, of the antibodyIMGN853, or an antibody described in US20120009181; U.S. Pat. No.4,851,332, LK26: U.S. Pat. No. 5,952,484.

In one embodiment, an antigen binding domain against ERBB2 (Her2/neu) isan antigen binding portion, e.g., CDRs, of the antibody trastuzumab, orpertuzumab.

In one embodiment, an antigen binding domain against MUC1 is an antigenbinding portion, e.g., CDRs, of the antibody SAR566658.

In one embodiment, the antigen binding domain against EGFR is antigenbinding portion, e.g., CDRs, of the antibody cetuximab, panitumumab,zalutumumab, nimotuzumab, or matuzumab.

In one embodiment, an antigen binding domain against NCAM is an antigenbinding portion, e.g., CDRs, of the antibody clone 2-2B: MAB5324 (EMDMillipore)

In one embodiment, an antigen binding domain against Ephrin B2 is anantigen binding portion, e.g., CDRs, of an antibody described in, e.g.,Abengozar et al., Blood 119(19):4565-4576 (2012).

In one embodiment, an antigen binding domain against IGF-I receptor isan antigen binding portion, e.g., CDRs, of an antibody described in,e.g., U.S. Pat. No. 8,344,112 B2; EP2322550 A1; WO 2006/138315, orPCT/US2006/022995.

In one embodiment, an antigen binding domain against CAIX is an antigenbinding portion, e.g., CDRs, of the antibody clone 303123 (R&D Systems).

In one embodiment, an antigen binding domain against LMP2 is an antigenbinding portion, e.g., CDRs, of an antibody described in, e.g., U.S.Pat. No. 7,410,640, or US20050129701.

In one embodiment, an antigen binding domain against gp100 is an antigenbinding portion, e.g., CDRs, of the antibody HMB45, NKIbetaB, or anantibody described in WO2013165940, or US20130295007

In one embodiment, an antigen binding domain against tyrosinase is anantigen binding portion, e.g., CDRs, of an antibody described in, e.g.,U.S. Pat. No. 5,843,674; or US 19950504048.

In one embodiment, an antigen binding domain against EphA2 is an antigenbinding portion, e.g., CDRs, of an antibody described in, e.g., Yu etal., Mol Ther 22(1):102-111 (2014).

In one embodiment, an antigen binding domain against GD3 is an antigenbinding portion, e.g., CDRs, of an antibody described in, e.g., U.S.Pat. Nos. 7,253,263; 8,207,308; US 20120276046; EP1013761 A3;20120276046; WO2005035577; or U.S. Pat. No. 6,437,098.

In one embodiment, an antigen binding domain against fucosyl GM1 is anantigen binding portion, e.g., CDRs, of an antibody described in, e.g.,US20100297138; or WO2007/067992.

In one embodiment, an antigen binding domain against sLe is an antigenbinding portion, e.g., CDRs, of the antibody G193 (for lewis Y), seeScott A M et al, Cancer Res 60: 3254-61 (2000), also as described inNeeson et al, J Immunol May 2013 190 (Meeting Abstract Supplement)177.10.

In one embodiment, an antigen binding domain against GM3 is an antigenbinding portion, e.g., CDRs, of the antibody CA 2523449 (mAb 14F7).

In one embodiment, an antigen binding domain against HMWMAA is anantigen binding portion, e.g., CDRs, of an antibody described in, e.g.,Kmiecik et al., Oncoimmunology 3(1):e27185 (2014) (PMID: 24575382)(mAb9.2.27); U.S. Pat. No. 6,528,481; WO2010033866; or US 20140004124.

In one embodiment, an antigen binding domain against o-acetyl-GD2 is anantigen binding portion, e.g., CDRs, of the antibody 8B6.

In one embodiment, an antigen binding domain against TEM1/CD248 is anantigen binding portion, e.g., CDRs, of an antibody described in, e.g.,Marty et al., Cancer Lett 235(2):298-308 (2006); Zhao et al., J ImmunolMethods 363(2):221-232 (2011).

In one embodiment, an antigen binding domain against CLDN6 is an antigenbinding portion, e.g., CDRs, of the antibody IMAB027 (GanymedPharmaceuticals), see e.g., clinicaltrial.gov/show/NCT02054351.

In one embodiment, an antigen binding domain against TSHR is an antigenbinding portion, e.g., CDRs, of an antibody described in, e.g., U.S.Pat. Nos. 8,603,466; 8,501,415; or U.S. Pat. No. 8,309,693.

In one embodiment, an antigen binding domain against GPRC5D is anantigen binding portion, e.g., CDRs, of the antibody FAB6300A (R&DSystems); or LS-A4180 (Lifespan Biosciences).

In one embodiment, an antigen binding domain against CD97 is an antigenbinding portion, e.g., CDRs, of an antibody described in, e.g., U.S.Pat. No. 6,846,911; de Groot et al., J Immunol 183(6):4127-4134 (2009);or an antibody from R&D:MAB3734.

In one embodiment, an antigen binding domain against ALK is an antigenbinding portion, e.g., CDRs, of an antibody described in, e.g.,Mino-Kenudson et al., Clin Cancer Res 16(5):1561-1571 (2010).

In one embodiment, an antigen binding domain against polysialic acid isan antigen binding portion, e.g., CDRs, of an antibody described in,e.g., Nagae et al., J Biol Chem 288(47):33784-33796 (2013)

In one embodiment, an antigen binding domain against PLAC1 is an antigenbinding portion, e.g., CDRs, of an antibody described in, e.g., Ghods etal., Biotechnol Appl Biochem 2013 doi:10.1002/bab.1177.

In one embodiment, an antigen binding domain against GloboH is anantigen binding portion of the antibody VK9; or an antibody describedin, e.g., Kudryashov V et al, Glycoconj J. 15(3):243-9 (1998), Lou etal., Proc Natl Acad Sci USA 111(7):2482-2487 (2014); MBr1: Bremer E-G etal. J Biol Chem 259:14773-14777 (1984).

In one embodiment, an antigen binding domain against NY-BR-1 is anantigen binding portion, e.g., CDRs of an antibody described in, e.g.,Jager et al., Appl Immunohistochem Mol Morphol 15(1):77-83 (2007).

In one embodiment, an antigen binding domain against WT-1 is an antigenbinding portion, e.g., CDRs, of an antibody described in, e.g., Dao etal., Sci Transl Med 5(176):176ra33 (2013); or WO2012/135854.

In one embodiment, an antigen binding domain against MAGE-A1 is anantigen binding portion, e.g., CDRs, of an antibody described in, e.g.,Willemsen et al., J Immunol 174(12):7853-7858 (2005) (TCR-like scFv).

In one embodiment, an antigen binding domain against sperm protein 17 isan antigen binding portion, e.g., CDRs, of an antibody described in,e.g., Song et al., Target Oncol 2013 Aug. 14 (PMID: 23943313); Song etal., Med Oncol 29(4):2923-2931 (2012).

In one embodiment, an antigen binding domain against Tie 2 is an antigenbinding portion, e.g., CDRs, of the antibody AB33 (Cell SignalingTechnology).

In one embodiment, an antigen binding domain against MAD-CT-2 is anantigen binding portion, e.g., CDRs, of an antibody described in, e.g.,PMID: 2450952; U.S. Pat. No. 7,635,753.

In one embodiment, an antigen binding domain against Fos-related antigen1 is an antigen binding portion, e.g., CDRs, of the antibody 12F9 (NovusBiologicals).

In one embodiment, an antigen binding domain against MelanA/MART1 is anantigen binding portion, e.g., CDRs, of an antibody described in,EP2514766 A2; or U.S. Pat. No. 7,749,719.

In one embodiment, an antigen binding domain against sarcomatranslocation breakpoints is an antigen binding portion, e.g., CDRs, ofan antibody described in, e.g., Luo et al, EMBO Mol. Med. 4(6):453-461(2012).

In one embodiment, an antigen binding domain against TRP-2 is an antigenbinding portion, e.g., CDRs, of an antibody described in, e.g., Wang etal, J Exp Med. 184(6):2207-16 (1996).

In one embodiment, an antigen binding domain against CYP1B1 is anantigen binding portion, e.g., CDRs, of an antibody described in, e.g.,Maecker et al, Blood 102 (9): 3287-3294 (2003).

In one embodiment, an antigen binding domain against RAGE-1 is anantigen binding portion, e.g., CDRs, of the antibody MAB5328 (EMDMillipore).

In one embodiment, an antigen binding domain against human telomerasereverse transcriptase is an antigen binding portion, e.g., CDRs, of theantibody cat no: LS-B95-100 (Lifespan Biosciences)

In one embodiment, an antigen binding domain against intestinal carboxylesterase is an antigen binding portion, e.g., CDRs, of the antibody4F12: cat no: LS-B6190-50 (Lifespan Biosciences).

In one embodiment, an antigen binding domain against mut hsp70-2 is anantigen binding portion, e.g., CDRs, of the antibody LifespanBiosciences: monoclonal: cat no: LS-C133261-100 (Lifespan Biosciences).

In one embodiment, an antigen binding domain against CD79a is an antigenbinding portion, e.g., CDRs, of the antibody Anti-CD79a antibody[HM47/A9] (ab3121), available from Abcam; antibody CD79A Antibody #3351available from Cell Signalling Technology; or antibodyHPA017748-Anti-CD79A antibody produced in rabbit, available from SigmaAldrich.

In one embodiment, an antigen binding domain against CD79b is an antigenbinding portion, e.g., CDRs, of the antibody polatuzumab vedotin,anti-CD79b described in Dornan et al., “Therapeutic potential of ananti-CD79b antibody-drug conjugate, anti-CD79b-vc-MMAE, for thetreatment of non-Hodgkin lymphoma” Blood. 2009 Sep. 24; 114(13):2721-9.doi: 10.1182/blood-2009-02-205500. Epub 2009 Jul. 24, or the bispecificantibody Anti-CD79b/CD3 described in “4507 Pre-Clinical Characterizationof T Cell-Dependent Bispecific Antibody Anti-CD79b/CD3 As a PotentialTherapy for B Cell Malignancies” Abstracts of 56th ASH Annual Meetingand Exposition, San Francisco, Calif. December 6-9 2014.

In one embodiment, an antigen binding domain against CD72 is an antigenbinding portion, e.g., CDRs, of the antibody J3-109 described in Myers,and Uckun, “An anti-CD72 immunotoxin against therapy-refractoryB-lineage acute lymphoblastic leukemia.” Leuk Lymphoma. 1995 June;18(1-2):119-22, or anti-CD72 (10D6.8.1, mIgG1) described in Polson etal., “Antibody-Drug Conjugates for the Treatment of Non-Hodgkin'sLymphoma: Target and Linker-Drug Selection” Cancer Res Mar. 15, 2009 69;2358.

In one embodiment, an antigen binding domain against LAIR1 is an antigenbinding portion, e.g., CDRs, of the antibody ANT-301 LAIR1 antibody,available from ProSpec; or anti-human CD305 (LAIR1) Antibody, availablefrom BioLegend.

In one embodiment, an antigen binding domain against FCAR is an antigenbinding portion, e.g., CDRs, of the antibody CD89/FCARAntibody (Catalog#10414-H08H), available from Sino Biological Inc.

In one embodiment, an antigen binding domain against LILRA2 is anantigen binding portion, e.g., CDRs, of the antibody LILRA2 monoclonalantibody (M17), clone 3C7, available from Abnova, or Mouse Anti-LILRA2antibody, Monoclonal (2D7), available from Lifespan Biosciences.

In one embodiment, an antigen binding domain against CD300LF is anantigen binding portion, e.g., CDRs, of the antibody MouseAnti-CMRF35-like molecule 1 antibody, Monoclonal[UP-D2], available fromBioLegend, or Rat Anti-CMRF35-like molecule 1 antibody,Monoclonal[234903], available from R&D Systems.

In one embodiment, an antigen binding domain against CLEC12A is anantigen binding portion, e.g., CDRs, of the antibody Bispecific T cellEngager (BiTE) scFv-antibody and ADC described in Noordhuis et al.,“Targeting of CLEC12A In Acute Myeloid Leukemia byAntibody-Drug-Conjugates and Bispecific CLL-1×CD3 BiTE Antibody” 53rdASH Annual Meeting and Exposition, Dec. 10-13, 2011, and MCLA-117(Merus).

In one embodiment, an antigen binding domain against BST2 (also calledCD317) is an antigen binding portion, e.g., CDRs, of the antibody MouseAnti-CD317 antibody, Monoclonal[3H4], available from Antibodies-Onlineor Mouse Anti-CD317 antibody, Monoclonal[696739], available from R&DSystems.

In one embodiment, an antigen binding domain against EMR2 (also calledCD312) is an antigen binding portion, e.g., CDRs, of the antibody MouseAnti-CD312 antibody, Monoclonal[LS-B8033] available from LifespanBiosciences, or Mouse Anti-CD312 antibody, Monoclonal[494025] availablefrom R&D Systems.

In one embodiment, an antigen binding domain against LY75 is an antigenbinding portion, e.g., CDRs, of the antibody Mouse Anti-Lymphocyteantigen 75 antibody, Monoclonal[HD30] available from EMD Millipore orMouse Anti-Lymphocyte antigen 75 antibody, Monoclonal[A15797] availablefrom Life Technologies.

In one embodiment, an antigen binding domain against GPC3 is an antigenbinding portion, e.g., CDRs, of the antibody hGC33 described in NakanoK, Ishiguro T, Konishi H, et al. Generation of a humanized anti-glypican3 antibody by CDR grafting and stability optimization. Anticancer Drugs.2010 November; 21(10):907-916, or MDX-1414, HN3, or YP7, all three ofwhich are described in Feng et al., “Glypican-3 antibodies: a newtherapeutic target for liver cancer.” FEBS Lett. 2014 Jan. 21;588(2):377-82.

In one embodiment, an antigen binding domain against FCRL5 is an antigenbinding portion, e.g., CDRs, of the anti-FcRL5 antibody described inElkins et al., “FcRL5 as a target of antibody-drug conjugates for thetreatment of multiple myeloma” Mol Cancer Ther. 2012 October;11(10):2222-32.

In one embodiment, an antigen binding domain against IGLL1 is an antigenbinding portion, e.g., CDRs, of the antibody Mouse Anti-Immunoglobulinlambda-like polypeptide 1 antibody, Monoclonal[AT1G4] available fromLifespan Biosciences, Mouse Anti-Immunoglobulin lambda-like polypeptide1 antibody, Monoclonal[HSL11] available from BioLegend.

In one embodiment, the antigen binding domain comprises one, two three(e.g., all three) heavy chain CDRs, HC CDR1, HC CDR2 and HC CDR3, froman antibody listed above, and/or one, two, three (e.g., all three) lightchain CDRs, LC CDR1, LC CDR2 and LC CDR3, from an antibody listed above.In one embodiment, the antigen binding domain comprises a heavy chainvariable region and/or a variable light chain region of an antibodylisted above.

In another aspect, the antigen binding domain comprises a humanizedantibody or an antibody fragment. In some aspects, a non-human antibodyis humanized, where specific sequences or regions of the antibody aremodified to increase similarity to an antibody naturally produced in ahuman or fragment thereof. In one aspect, the antigen binding domain ishumanized.

Bispecific CARs

In certain embodiments, the antigen binding domain is a bi- ormulti-specific molecule (e.g., a multispecific antibody molecule). In anembodiment a multispecific antibody molecule is a bispecific antibodymolecule. A bispecific antibody has specificity for no more than twoantigens. A bispecific antibody molecule is characterized by a firstimmunoglobulin variable domain sequence which has binding specificityfor a first epitope and a second immunoglobulin variable domain sequencethat has binding specificity for a second epitope. In an embodiment thefirst and second epitopes are on the same antigen, e.g., the sameprotein (or subunit of a multimeric protein). In an embodiment the firstand second epitopes overlap. In an embodiment the first and secondepitopes do not overlap. In an embodiment the first and second epitopesare on different antigens, e.g., different proteins (or differentsubunits of a multimeric protein). In an embodiment a bispecificantibody molecule comprises a heavy chain variable domain sequence and alight chain variable domain sequence which have binding specificity fora first epitope and a heavy chain variable domain sequence and a lightchain variable domain sequence which have binding specificity for asecond epitope. In an embodiment a bispecific antibody moleculecomprises a half antibody having binding specificity for a first epitopeand a half antibody having binding specificity for a second epitope. Inan embodiment a bispecific antibody molecule comprises a half antibody,or fragment thereof, having binding specificity for a first epitope anda half antibody, or fragment thereof, having binding specificity for asecond epitope. In an embodiment a bispecific antibody moleculecomprises a scFv, or fragment thereof, have binding specificity for afirst epitope and a scFv, or fragment thereof, have binding specificityfor a second epitope.

In certain embodiments, the antibody molecule is a multi-specific (e.g.,a bispecific or a trispecific) antibody molecule. Such molecules includebispecific fusion proteins, e.g., an expression construct containing twoscFvs with a hydrophilic helical peptide linker between them and a fullconstant region, as described in, e.g., U.S. Pat. No. 5,637,481;minibody constructs with linked VL and VH chains further connected withpeptide spacers to an antibody hinge region and CH3 region, which can bedimerized to form bispecific/multivalent molecules, as described in,e.g., U.S. Pat. No. 5,837,821; String of VH domains (or VL domains infamily members) connected by peptide linkages with crosslinkable groupsat the C-terminus futher associated with VL domains to form a series ofFVs (or scFvs), as described in, e.g., U.S. Pat. No. 5,864,019; andsingle chain binding polypeptides with both a VH and a VL domain linkedthrough a peptide linker are combined into multivalent structuresthrough non-covalent or chemical crosslinking to form, e.g.,homobivalent, heterobivalent, trivalent, and tetravalent structuresusing both scFV or diabody type format, as described in, e.g., U.S. Pat.No. 5,869,620. The contents of the above-referenced applications areincorporated herein by reference in their entireties.

Within each antibody or antibody fragment (e.g., scFv) of a bispecificantibody molecule, the VH can be upstream or downstream of the VL. Insome embodiments, the upstream antibody or antibody fragment (e.g.,scFv) is arranged with its VH (VH₁) upstream of its VL (VL₁) and thedownstream antibody or antibody fragment (e.g., scFv) is arranged withits VL (VL₂) upstream of its VH (VH₂), such that the overall bispecificantibody molecule has the arrangement VH₁-VL₁-VL₂-VH₂. In otherembodiments, the upstream antibody or antibody fragment (e.g., scFv) isarranged with its VL (VL₁) upstream of its VH (VH₁) and the downstreamantibody or antibody fragment (e.g., scFv) is arranged with its VH (VH₂)upstream of its VL (VL₂), such that the overall bispecific antibodymolecule has the arrangement VL₁-VH₁-VH₂-VL₂. Optionally, a linker isdisposed between the two antibodies or antibody fragments (e.g., scFvs),e.g., between VL₁ and VL₂ if the construct is arranged asVH₁-VL₁-VL₂-VH₂, or between VH₁ and VH₂ if the construct is arranged asVL₁-VH₁-VH₂-VL₂. The linker may be a linker as described herein, e.g., a(Gly₄-Ser)n linker, wherein n is 1, 2, 3, 4, 5, or 6, preferably 4 (SEQID NO: 29). In general, the linker between the two scFvs should be longenough to avoid mispairing between the domains of the two scFvs.Optionally, a linker is disposed between the VL and VH of the firstscFv. Optionally, a linker is disposed between the VL and VH of thesecond scFv. In constructs that have multiple linkers, any two or moreof the linkers can be the same or different. Accordingly, in someembodiments, a bispecific CAR comprises VLs, VHs, and optionally one ormore linkers in an arrangement as described herein.

Transmembrane Domain

A transmembrane domain of the CAR may be any polypeptide domain capableof traversing a phospholipid bilayer, so that one end of the domain isattached to, e.g., an extracellular antigen binding domain, and theother end is, e.g., attached to an intracellular signaling domain. Atransmembrane domain can include one or more additional amino acidsadjacent to the transmembrane region, e.g., one or more amino acidsassociated with the extracellular region of the protein from which thetransmembrane was derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15amino acids of the extracellular region) and/or one or more additionalamino acids associated with the intracellular region of the protein fromwhich the transmembrane protein is derived (e.g., 1, 2, 3, 4, 5, 6, 7,8, 9, 10 up to 15 amino acids of the intracellular region). Thetransmembrane domain can be is associated with one of the other domainsof the CAR. In some instances, the transmembrane domain can be selectedor modified by amino acid substitution to avoid binding of such domainsto the transmembrane domains of the same or different surface membraneproteins, e.g., to minimize interactions with other members of thereceptor complex. In one aspect, the transmembrane domain is capable ofhomodimerization with another CAR on the surface of a CAR-expressingcell. In a different aspect, the amino acid sequence of thetransmembrane domain may be modified or substituted so as to minimizeinteractions with the binding domains of the native binding partnerpresent in the same CAR-expressing cell. A transmembrane domain ofparticular use in this invention may include at least the transmembraneregion(s) of e.g., the alpha, beta or zeta chain of the T-cell receptor,CD28, CD27, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33,CD37, CD64, CD80, CD86, CD134, CD137, CD154. In some embodiments, atransmembrane domain may include at least the transmembrane region(s)of, e.g., KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278),4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1),NKp44, NKp30, NKp46, CD160, CD19, IL2R beta, IL2R gamma, IL7R a, ITGA1,VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d,ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11 b, ITGAX, CD11c, ITGB1,CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244,2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55),PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150,IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG/Cbp, NKG2D, NKG2C.

In some instances, the transmembrane domain can be attached to theextracellular region of the CAR, e.g., the antigen binding domain of theCAR, via a hinge, e.g., a hinge from a human protein. For example, inone embodiment, the hinge can be a human Ig (immunoglobulin) hinge(e.g., an IgG4 hinge, an IgD hinge), a GS linker (e.g., a GS linkerdescribed herein), a KIR2DS2 hinge or a CD8a hinge. In one embodiment,the hinge or spacer comprises (e.g., consists of) the amino acidsequence of SEQ ID NO:4. In one aspect, the transmembrane domaincomprises (e.g., consists of) a transmembrane domain of SEQ ID NO: 12.

In certain embodiments, the encoded transmembrane domain comprises anamino acid sequence of a CD8 transmembrane domain having at least one,two or three modifications but not more than 20, 10 or 5 modificationsof an amino acid sequence of SEQ ID NO: 12, or a sequence with 95-99%identity to an amino acid sequence of SEQ ID NO: 12. In one embodiment,the encoded transmembrane domain comprises the sequence of SEQ ID NO:12.

In other embodiments, the nucleic acid molecule encoding the CARcomprises a nucleotide sequence of a CD8 transmembrane domain, e.g.,comprising the sequence of SEQ ID NO: 13, or a sequence with 95-99%identity thereof.

In certain embodiments, the encoded antigen binding domain is connectedto the transmembrane domain by a hinge region. In one embodiment, theencoded hinge region comprises the amino acid sequence of a CD8 hinge,e.g., SEQ ID NO: 4; or the amino acid sequence of an IgG4 hinge, e.g.,SEQ ID NO: 6, or a sequence with 95-99% identity to SEQ ID NO:4 or 6. Inother embodiments, the nucleic acid sequence encoding the hinge regioncomprises a sequence of SEQ ID NO: 5 or SEQ ID NO: 7, corresponding to aCD8 hinge or an IgG4 hinge, respectively, or a sequence with 95-99%identity to SEQ ID NO:5 or 7.

In one aspect, the hinge or spacer comprises an IgG4 hinge. For example,in one embodiment, the hinge or spacer comprises a hinge of the aminoacid sequenceESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKM (SEQ ID NO:6). In some embodiments, the hinge orspacer comprises a hinge encoded by a nucleotide sequence of

(SEQ ID NO: 7) GAGAGCAAGTACGGCCCTCCCTGCCCCCCTTGCCCTGCCCCCGAGTTCCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAGGTGACCTGTGTGGTGGTGGACGTGTCCCAGGAGGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCCGGGAGGAGCAGTTCAATAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGTAAGGTGTCCAACAAGGGCCTGCCCAGCAGCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTCGGGAGCCCCAGGTGTACACCCTGCCCCCTAGCCAAGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCCGGCTGACCGTGGACAAGAGCCGGTGGCAGGAGGGCAACGTCTTTAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCTGGGCAAGATG.

In one aspect, the hinge or spacer comprises an IgD hinge. For example,in one embodiment, the hinge or spacer comprises a hinge of the aminoacid sequenceRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDH (SEQ ID NO:8). In some embodiments, the hinge or spacercomprises a hinge encoded by a nucleotide sequence of

(SEQ ID NO: 9) AGGTGGCCCGAAAGTCCCAAGGCCCAGGCATCTAGTGTTCCTACTGCACAGCCCCAGGCAGAAGGCAGCCTAGCCAAAGCTACTACTGCACCTGCCACTACGCGCAATACTGGCCGTGGCGGGGAGGAGAAGAAAAAGGAGAAAGAGAAAGAAGAACAGGAAGAGAGGGAGACCAAGACCCCTGAATGTCCATCCCATACCCAGCCGCTGGGCGTCTATCTCTTGACTCCCGCAGTACAGGACTTGTGGCTTAGAGATAAGGCCACCTTTACATGTTTCGTCGTGGGCTCTGACCTGAAGGATGCCCATTTGACTTGGGAGGTTGCCGGAAAGGTACCCACAGGGGGGGTTGAGGAAGGGTTGCTGGAGCGCCATTCCAATGGCTCTCAGAGCCAGCACTCAAGACTCACCCTTCCGAGATCCCTGTGGAACGCCGGGACCTCTGTCACATGTACTCTAAATCATCCTAGCCTGCCCCCACAGCGTCTGATGGCCCTTAGAGAGCCAGCCGCCCAGGCACCAGTTAAGCTTAGCCTGAATCTGCTCGCCAGTAGTGATCCCCCAGAGGCCGCCAGCTGGCTCTTATGCGAAGTGTCCGGCTTTAGCCCGCCCAACATCTTGCTCATGTGGCTGGAGGACCAGCGAGAAGTGAACACCAGCGGCTTCGCTCCAGCCCGGCCCCCACCCCAGCCGGGTTCTACCACATTCTGGGCCTGGAGTGTCTTAAGGGTCCCAGCACCACCTAGCCCCCAGCCAGCCACATACACCTGTGTTGTGTCCCATGAAGATAGCAGGACCCTGCTAAATGCTTCTAGGAGTCTGGAGGTTTCCTACGTGACTGACCATT.

In one aspect, the transmembrane domain may be recombinant, in whichcase it will comprise predominantly hydrophobic residues such as leucineand valine. In one aspect a triplet of phenylalanine, tryptophan andvaline can be found at each end of a recombinant transmembrane domain.

Optionally, a short oligo- or polypeptide linker, between 2 and 10 aminoacids in length may form the linkage between the transmembrane domainand the cytoplasmic region of the CAR. A glycine-serine doublet providesa particularly suitable linker. For example, in one aspect, the linkercomprises the amino acid sequence of GGGGSGGGGS (SEQ ID NO: 10). In someembodiments, the linker is encoded by a nucleotide sequence ofGGTGGCGGAGGTTCTGGAGGTGGAGGTTCC (SEQ ID NO: 11).

In one aspect, the hinge or spacer comprises a KIR2DS2 hinge.

Signaling Domains

A CAR may include one or more intracellular signaling domains, which mayactivate and/or inhibit one or more intracellular signaling pathways inresponse to binding of the extracellular antigen binding domain to aligand. In some instances, the intracellular signaling domain may becapable of inducing an immune response. For example, the intracellularsignaling domain of a CAR expressed by a T cell may induce activation ofthe T cell upon binding of the antigen binding domain to its cognatetarget molecule of interest.

In embodiments of the invention having an intracellular signalingdomain, such a domain can contain, e.g., one or more of a primarysignaling domain and/or a costimulatory signaling domain. In someembodiments, the intracellular signaling domain comprises a sequenceencoding a primary signaling domain. In some embodiments, theintracellular signaling domain comprises a costimulatory signalingdomain. In some embodiments, the intracellular signaling domaincomprises a a primary signaling domain and a costimulatory signalingdomain.

The intracellular signaling sequences within the cytoplasmic portion ofthe CAR of the invention may be linked to each other in a random orspecified order. Optionally, a short oligo- or polypeptide linker, forexample, between 2 and 10 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or10 amino acids) in length may form the linkage between intracellularsignaling sequences. In one embodiment, a glycine-serine doublet can beused as a suitable linker. In one embodiment, a single amino acid, e.g.,an alanine, a glycine, can be used as a suitable linker.

In one aspect, the intracellular signaling domain is designed tocomprise two or more, e.g., 2, 3, 4, 5, or more, costimulatory signalingdomains. In an embodiment, the two or more, e.g., 2, 3, 4, 5, or more,costimulatory signaling domains, are separated by a linker molecule,e.g., a linker molecule described herein. In one embodiment, theintracellular signaling domain comprises two costimulatory signalingdomains. In some embodiments, the linker molecule is a glycine residue.In some embodiments, the linker is an alanine residue.

Primary Signaling Domains

A primary signaling domain regulates primary activation of the TCRcomplex either in a stimulatory way, or in an inhibitory way. Primaryintracellular signaling domains that act in a stimulatory manner maycontain signaling motifs which are known as immunoreceptortyrosine-based activation motifs or ITAMs.

Examples of ITAM containing primary intracellular signaling domains thatare of particular use in the invention include those of CD3 zeta, commonFcR gamma (FCER1G), Fc gamma RIIa, FcR beta (Fc Epsilon R1 b), CD3gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, and DAP12. In oneembodiment, a CAR of the invention comprises an intracellular signalingdomain, e.g., a primary signaling domain of CD3-zeta.

In one embodiment, the encoded primary signaling domain comprises afunctional signaling domain of CD3 zeta. The encoded CD3 zeta primarysignaling domain can comprise an amino acid sequence having at leastone, two or three modifications but not more than 20, 10 or 5modifications of an amino acid sequence of SEQ ID NO: 18 or SEQ ID NO:20, or a sequence with 95-99% identity to an amino acid sequence of SEQID NO: 18 or SEQ ID NO: 20. In some embodiments, the encoded primarysignaling domain comprises a sequence of SEQ ID NO: 18 or SEQ ID NO: 20.In other embodiments, the nucleic acid sequence encoding the primarysignaling domain comprises a sequence of SEQ ID NO: 19 or SEQ ID NO: 21,or a sequence with 95-99% identity thereof.

Costimulatory Signaling Domain

In some embodiments, the encoded intracellular signaling domaincomprises a a costimulatory signaling domain. For example, theintracellular signaling domain can comprise a primary signaling domainand a costimulatory signaling domain. In some embodiments, the encodedcostimulatory signaling domain comprises a functional signaling domainof a protein chosen from one or more of CD27, CD28, 4-1 BB (CD137),OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1(LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically bindswith CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80(KLRF1), CD160, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma,IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f,ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11 b, ITGAX,CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE/RANKL,DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1,CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6(NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG(CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, NKp44, NKp30, NKp46, orNKG2D.

In certain embodiments, the encoded costimulatory signaling domaincomprises an amino acid sequence having at least one, two or threemodifications but not more than 20, 10 or 5 modifications of an aminoacid sequence of SEQ ID NO: 14 or SEQ ID NO: 16, or a sequence with95-99% identity to an amino acid sequence of SEQ ID NO:14 or SEQ ID NO:16. In one embodiment, the encoded costimulatory signaling domaincomprises a sequence of SEQ ID NO: 14 or SEQ ID NO: 16. In otherembodiments, the nucleic acid sequence encoding the costimulatorysignaling domain comprises a sequence of SEQ ID NO: 15 or SEQ ID NO: 17,or a sequence with 95-99% identity thereof.

In other embodiments, the encoded intracellular domain comprises thesequence of SEQ ID NO: 14 or SEQ ID NO: 16, and the sequence of SEQ IDNO: 18 or SEQ ID NO: 20, wherein the sequences comprising theintracellular signaling domain are expressed in the same frame and as asingle polypeptide chain.

In certain embodiments, the nucleic acid sequence encoding theintracellular signaling domain comprises a sequence of SEQ ID NO: 15 orSEQ ID NO: 17, or a sequence with 95-99% identity thereof, and asequence of SEQ ID NO: 19 or SEQ ID NO: 21, or a sequence with 95-99%identity thereof.

In some embodiments, the nucleic acid molecule further encodes a leadersequence. In one embodiment, the leader sequence comprises the sequenceof SEQ ID NO: 2.

In one aspect, the intracellular signaling domain is designed tocomprise the signaling domain of CD3-zeta and the signaling domain ofCD28. In one aspect, the intracellular signaling domain is designed tocomprise the signaling domain of CD3-zeta and the signaling domain of4-1 BB. In one aspect, the signaling domain of 4-1 BB is a signalingdomain of SEQ ID NO: 14. In one aspect, the signaling domain of CD3-zetais a signaling domain of SEQ ID NO: 18.

In one aspect, the intracellular signaling domain is designed tocomprise the signaling domain of CD3-zeta and the signaling domain ofCD27. In one aspect, the signaling domain of CD27 comprises an aminoacid sequence of QRRKYRSNKGESPVEPAEPCRYSCPREEEGSTIPIQEDYRKPEPACSP (SEQID NO: 16). In one aspect, the signaling domain of CD27 is encoded by anucleic acid sequence of

(SEQ ID NO: 17) AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC.

Host Cells for CAR Expression

As noted above, in some aspects the invention pertains to a cell, e.g.,an immune effector cell, (e.g., a population of cells, e.g., apopulation of immune effector cells) comprising a nucleic acid molecule,a CAR polypeptide molecule, or a vector as described herein.

In certain aspects of the present disclosure, immune effector cells,e.g., T cells, can be obtained from a unit of blood collected from asubject using any number of techniques known to the skilled artisan,such as Ficoll™ separation. In one preferred aspect, cells from thecirculating blood of an individual are obtained by apheresis. Theapheresis product typically contains lymphocytes, including T cells,monocytes, granulocytes, B cells, other nucleated white blood cells, redblood cells, and platelets. In one aspect, the cells collected byapheresis may be washed to remove the plasma fraction and, optionally,to place the cells in an appropriate buffer or media for subsequentprocessing steps. In one embodiment, the cells are washed with phosphatebuffered saline (PBS). In an alternative embodiment, the wash solutionlacks calcium and may lack magnesium or may lack many if not alldivalent cations.

Initial activation steps in the absence of calcium can lead to magnifiedactivation. As those of ordinary skill in the art would readilyappreciate a washing step may be accomplished by methods known to thosein the art, such as by using a semi-automated “flow-through” centrifuge(for example, the Cobe 2991 cell processor, the Baxter CytoMate, or theHaemonetics Cell Saver 5) according to the manufacturer's instructions.After washing, the cells may be resuspended in a variety ofbiocompatible buffers, such as, for example, Ca-free, Mg-free PBS,PlasmaLyte A, or other saline solution with or without buffer.Alternatively, the undesirable components of the apheresis sample may beremoved and the cells directly resuspended in culture media.

It is recognized that the methods of the application can utilize culturemedia conditions comprising 5% or less, for example 2%, human AB serum,and employ known culture media conditions and compositions, for examplethose described in Smith et al., “Ex vivo expansion of human T cells foradoptive immunotherapy using the novel Xeno-free CTS Immune Cell SerumReplacement” Clinical & Translational Immunology (2015) 4, e31;doi:10.1038/cti.2014.31.

In one aspect, T cells are isolated from peripheral blood lymphocytes bylysing the red blood cells and depleting the monocytes, for example, bycentrifugation through a PERCOLL™ gradient or by counterflow centrifugalelutriation.

The methods described herein can include, e.g., selection of a specificsubpopulation of immune effector cells, e.g., T cells, that are a Tregulatory cell-depleted population, CD25+ depleted cells, using, e.g.,a negative selection technique, e.g., described herein. Preferably, thepopulation of T regulatory depleted cells contains less than 30%, 25%,20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% of CD25+ cells.

In one embodiment, T regulatory cells, e.g., CD25+ T cells, are removedfrom the population using an anti-CD25 antibody, or fragment thereof, ora CD25-binding ligand, IL-2. In one embodiment, the anti-CD25 antibody,or fragment thereof, or CD25-binding ligand is conjugated to asubstrate, e.g., a bead, or is otherwise coated on a substrate, e.g., abead. In one embodiment, the anti-CD25 antibody, or fragment thereof, isconjugated to a substrate as described herein.

In one embodiment, the T regulatory cells, e.g., CD25+ T cells, areremoved from the population using CD25 depletion reagent from Miltenyi™.In one embodiment, the ratio of cells to CD25 depletion reagent is 1e7cells to 20 uL, or 1e7 cells to 15 uL, or 1e7 cells to 10 uL, or 1e7cells to 5 uL, or 1e7 cells to 2.5 uL, or 1e7 cells to 1.25 uL. In oneembodiment, e.g., for T regulatory cells, e.g., CD25+ depletion, greaterthan 500 million cells/ml is used. In a further aspect, a concentrationof cells of 600, 700, 800, or 900 million cells/ml is used.

In one embodiment, the population of immune effector cells to bedepleted includes about 6×10⁹ CD25+ T cells. In other aspects, thepopulation of immune effector cells to be depleted include about 1×10⁹to 1×10¹⁰ CD25+ T cell, and any integer value in between. In oneembodiment, the resulting population T regulatory depleted cells has2×10⁹T regulatory cells, e.g., CD25+ cells, or less (e.g., 1×10⁹, 5×10⁸,1×10⁸, 5×10⁷, 1×10⁷, or less CD25+ cells).

In one embodiment, the T regulatory cells, e.g., CD25+ cells, areremoved from the population using the CliniMAC system with a depletiontubing set, such as, e.g., tubing 162-01. In one embodiment, theCliniMAC system is run on a depletion setting such as, e.g.,DEPLETION2.1.

Without wishing to be bound by a particular theory, decreasing the levelof negative regulators of immune cells (e.g., decreasing the number ofunwanted immune cells, e.g., T_(REG) cells), in a subject prior toapheresis or during manufacturing of a CAR-expressing cell product canreduce the risk of subject relapse. For example, methods of depletingT_(REG) cells are known in the art. Methods of decreasing T_(REG) cellsinclude, but are not limited to, cyclophosphamide, anti-GITR antibody(an anti-GITR antibody described herein), CD25-depletion, andcombinations thereof.

In some embodiments, the manufacturing methods comprise reducing thenumber of (e.g., depleting) T_(REG) cells prior to manufacturing of theCAR-expressing cell. For example, manufacturing methods comprisecontacting the sample, e.g., the apheresis sample, with an anti-GITRantibody and/or an anti-CD25 antibody (or fragment thereof, or aCD25-binding ligand), e.g., to deplete T_(REG) cells prior tomanufacturing of the CAR-expressing cell (e.g., T cell, NK cell)product.

In an embodiment, a subject is pre-treated with one or more therapiesthat reduce T_(REG) cells prior to collection of cells forCAR-expressing cell product manufacturing, thereby reducing the risk ofsubject relapse to CAR-expressing cell treatment. In an embodiment,methods of decreasing T_(REG) cells include, but are not limited to,administration to the subject of one or more of cyclophosphamide,anti-GITR antibody, CD25-depletion, or a combination thereof.Administration of one or more of cyclophosphamide, anti-GITR antibody,CD25-depletion, or a combination thereof, can occur before, during orafter an infusion of the CAR-expressing cell product.

In an embodiment, a subject is pre-treated with cyclophosphamide priorto collection of cells for CAR-expressing cell product manufacturing,thereby reducing the risk of subject relapse to CAR-expressing celltreatment. In an embodiment, a subject is pre-treated with an anti-GITRantibody prior to collection of cells for CAR-expressing cell productmanufacturing, thereby reducing the risk of subject relapse toCAR-expressing cell treatment.

In one embodiment, the population of cells to be removed are neither theregulatory T cells or tumor cells, but cells that otherwise negativelyaffect the expansion and/or function of CART cells, e.g. cellsexpressing CD14, CD11b, CD33, CD15, or other markers expressed bypotentially immune suppressive cells. In one embodiment, such cells areenvisioned to be removed concurrently with regulatory T cells and/ortumor cells, or following said depletion, or in another order.

The methods described herein can include more than one selection step,e.g., more than one depletion step. Enrichment of a T cell population bynegative selection can be accomplished, e.g., with a combination ofantibodies directed to surface markers unique to the negatively selectedcells. One method is cell sorting and/or selection via negative magneticimmunoadherence or flow cytometry that uses a cocktail of monoclonalantibodies directed to cell surface markers present on the cellsnegatively selected. For example, to enrich for CD4+ cells by negativeselection, a monoclonal antibody cocktail can include antibodies toCD14, CD20, CD11 b, CD16, HLA-DR, and CD8.

The methods described herein can further include removing cells from thepopulation which express a tumor antigen, e.g., a tumor antigen thatdoes not comprise CD25, e.g., CD19, CD30, CD38, CD123, CD20, CD14 orCD11 b, to thereby provide a population of T regulatory depleted, e.g.,CD25+ depleted, and tumor antigen depleted cells that are suitable forexpression of a CAR, e.g., a CAR described herein. In one embodiment,tumor antigen expressing cells are removed simultaneously with the Tregulatory, e.g., CD25+ cells. For example, an anti-CD25 antibody, orfragment thereof, and an anti-tumor antigen antibody, or fragmentthereof, can be attached to the same substrate, e.g., bead, which can beused to remove the cells or an anti-CD25 antibody, or fragment thereof,or the anti-tumor antigen antibody, or fragment thereof, can be attachedto separate beads, a mixture of which can be used to remove the cells.In other embodiments, the removal of T regulatory cells, e.g., CD25+cells, and the removal of the tumor antigen expressing cells issequential, and can occur, e.g., in either order.

Also provided are methods that include removing cells from thepopulation which express a check point inhibitor, e.g., a check pointinhibitor described herein, e.g., one or more of PD1+ cells, LAG3+cells, and TIM3+ cells, to thereby provide a population of T regulatorydepleted, e.g., CD25+ depleted cells, and check point inhibitor depletedcells, e.g., PD1+, LAG3+ and/or TIM3+ depleted cells. Exemplary checkpoint inhibitors include B7-H1, B7-1, CD160, P1H, 2B4, PD1, TIM3, CEACAM(e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, TIGIT, CTLA-4, BTLAand LAIR1. In one embodiment, check point inhibitor expressing cells areremoved simultaneously with the T regulatory, e.g., CD25+ cells. Forexample, an anti-CD25 antibody, or fragment thereof, and an anti-checkpoint inhibitor antibody, or fragment thereof, can be attached to thesame bead which can be used to remove the cells, or an anti-CD25antibody, or fragment thereof, and the anti-check point inhibitorantibody, or fragment there, can be attached to separate beads, amixture of which can be used to remove the cells. In other embodiments,the removal of T regulatory cells, e.g., CD25+ cells, and the removal ofthe check point inhibitor expressing cells is sequential, and can occur,e.g., in either order.

Methods described herein can include a positive selection step. Forexample, T cells can isolated by incubation with anti-CD3/anti-CD28(e.g., 3×28)-conjugated beads, such as DYNABEADS® M-450 CD3/CD28 T, fora time period sufficient for positive selection of the desired T cells.In one embodiment, the time period is about 30 minutes. In a furtherembodiment, the time period ranges from 30 minutes to 36 hours or longerand all integer values there between. In a further embodiment, the timeperiod is at least 1, 2, 3, 4, 5, or 6 hours. In yet another embodiment,the time period is 10 to 24 hours, e.g., 24 hours. Longer incubationtimes may be used to isolate T cells in any situation where there arefew T cells as compared to other cell types, such in isolating tumorinfiltrating lymphocytes (TIL) from tumor tissue or fromimmunocompromised individuals. Further, use of longer incubation timescan increase the efficiency of capture of CD8+ T cells. Thus, by simplyshortening or lengthening the time T cells are allowed to bind to theCD3/CD28 beads and/or by increasing or decreasing the ratio of beads toT cells (as described further herein), subpopulations of T cells can bepreferentially selected for or against at culture initiation or at othertime points during the process. Additionally, by increasing ordecreasing the ratio of anti-CD3 and/or anti-CD28 antibodies on thebeads or other surface, subpopulations of T cells can be preferentiallyselected for or against at culture initiation or at other desired timepoints.

In one embodiment, a T cell population can be selected that expressesone or more of IFN-γ, TNFα, IL-17A, IL-2, IL-3, IL-4, GM-CSF, IL-10,IL-13, granzyme B, and perforin, or other appropriate molecules, e.g.,other cytokines. Methods for screening for cell expression can bedetermined, e.g., by the methods described in PCT Publication No.: WO2013/126712.

For isolation of a desired population of cells by positive or negativeselection, the concentration of cells and surface (e.g., particles suchas beads) can be varied. In certain aspects, it may be desirable tosignificantly decrease the volume in which beads and cells are mixedtogether (e.g., increase the concentration of cells), to ensure maximumcontact of cells and beads. For example, in one aspect, a concentrationof 10 billion cells/ml, 9 billion/ml, 8 billion/ml, 7 billion/ml, 6billion/ml, or 5 billion/ml is used. In one aspect, a concentration of 1billion cells/ml is used. In yet one aspect, a concentration of cellsfrom 75, 80, 85, 90, 95, or 100 million cells/ml is used. In furtheraspects, concentrations of 125 or 150 million cells/ml can be used.

Using high concentrations can result in increased cell yield, cellactivation, and cell expansion. Further, use of high cell concentrationsallows more efficient capture of cells that may weakly express targetantigens of interest, such as CD28-negative T cells, or from sampleswhere there are many tumor cells present (e.g., leukemic blood, tumortissue, etc.). Such populations of cells may have therapeutic value andwould be desirable to obtain. For example, using high concentration ofcells allows more efficient selection of CD8+ T cells that normally haveweaker CD28 expression.

In a related aspect, it may be desirable to use lower concentrations ofcells. By significantly diluting the mixture of T cells and surface(e.g., particles such as beads), interactions between the particles andcells is minimized. This selects for cells that express high amounts ofdesired antigens to be bound to the particles. For example, CD4+ T cellsexpress higher levels of CD28 and are more efficiently captured thanCD8+ T cells in dilute concentrations. In one aspect, the concentrationof cells used is 5×10⁶/ml. In other aspects, the concentration used canbe from about 1×10⁵/ml to 1×10⁶/ml, and any integer value in between.

In other aspects, the cells may be incubated on a rotator for varyinglengths of time at varying speeds at either 2-10° C. or at roomtemperature.

T cells for stimulation can also be frozen after a washing step. Wishingnot to be bound by theory, the freeze and subsequent thaw step providesa more uniform product by removing granulocytes and to some extentmonocytes in the cell population. After the washing step that removesplasma and platelets, the cells may be suspended in a freezing solution.While many freezing solutions and parameters are known in the art andwill be useful in this context, one method involves using PBS containing20% DMSO and 8% human serum albumin, or culture media containing 10%Dextran 40 and 5% Dextrose, 20% Human Serum Albumin and 7.5% DMSO, or31.25% Plasmalyte-A, 31.25% Dextrose 5%, 0.45% NaCl, 10% Dextran 40 and5% Dextrose, 20% Human Serum Albumin, and 7.5% DMSO or other suitablecell freezing media containing for example, Hespan and PlasmaLyte A, thecells then are frozen to −80° C. at a rate of 1° per minute and storedin the vapor phase of a liquid nitrogen storage tank. Other methods ofcontrolled freezing may be used as well as uncontrolled freezingimmediately at −20° C. or in liquid nitrogen.

In certain aspects, cryopreserved cells are thawed and washed asdescribed herein and allowed to rest for one hour at room temperatureprior to activation using the methods of the present invention.

Also contemplated in the context of the invention is the collection ofblood samples or apheresis product from a subject at a time period priorto when the expanded cells as described herein might be needed. As such,the source of the cells to be expanded can be collected at any timepoint necessary, and desired cells, such as T cells, isolated and frozenfor later use in immune effector cell therapy for any number of diseasesor conditions that would benefit from immune effector cell therapy, suchas those described herein. In one aspect a blood sample or an apheresisis taken from a generally healthy subject. In certain aspects, a bloodsample or an apheresis is taken from a generally healthy subject who isat risk of developing a disease, but who has not yet developed adisease, and the cells of interest are isolated and frozen for lateruse. In certain aspects, the T cells may be expanded, frozen, and usedat a later time. In certain aspects, samples are collected from apatient shortly after diagnosis of a particular disease as describedherein but prior to any treatments. In a further aspect, the cells areisolated from a blood sample or an apheresis from a subject prior to anynumber of relevant treatment modalities, including but not limited totreatment with agents such as natalizumab, efalizumab, antiviral agents,chemotherapy, radiation, immunosuppressive agents, such as cyclosporin,azathioprine, methotrexate, mycophenolate, and FK506, antibodies, orother immunoablative agents such as CAMPATH, anti-CD3 antibodies,cytoxan, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid,steroids, FR901228, and irradiation.

In a further aspect of the present invention, T cells are obtained froma patient directly following treatment that leaves the subject withfunctional T cells. In this regard, it has been observed that followingcertain cancer treatments, in particular treatments with drugs thatdamage the immune system, shortly after treatment during the period whenpatients would normally be recovering from the treatment, the quality ofT cells obtained may be optimal or improved for their ability to expandex vivo. Likewise, following ex vivo manipulation using the methodsdescribed herein, these cells may be in a preferred state for enhancedengraftment and in vivo expansion. Thus, it is contemplated within thecontext of the present invention to collect blood cells, including Tcells, dendritic cells, or other cells of the hematopoietic lineage,during this recovery phase. Further, in certain aspects, mobilization(for example, mobilization with GM-CSF) and conditioning regimens can beused to create a condition in a subject wherein repopulation,recirculation, regeneration, and/or expansion of particular cell typesis favored, especially during a defined window of time followingtherapy. Illustrative cell types include T cells, B cells, dendriticcells, and other cells of the immune system.

In one embodiment, the immune effector cells expressing a CAR molecule,e.g., a CAR molecule described herein, are obtained from a subject thathas received a low, immune enhancing dose of an mTOR inhibitor. In anembodiment, the population of immune effector cells, e.g., T cells, tobe engineered to express a CAR, are harvested after a sufficient time,or after sufficient dosing of the low, immune enhancing, dose of an mTORinhibitor, such that the level of PD1 negative immune effector cells,e.g., T cells, or the ratio of PD1 negative immune effector cells, e.g.,T cells/PD1 positive immune effector cells, e.g., T cells, in thesubject or harvested from the subject has been, at least transiently,increased.

In other embodiments, population of immune effector cells, e.g., Tcells, which have, or will be engineered to express a CAR, can betreated ex vivo by contact with an amount of an mTOR inhibitor thatincreases the number of PD1 negative immune effector cells, e.g., Tcells or increases the ratio of PD1 negative immune effector cells,e.g., T cells/PD1 positive immune effector cells, e.g., T cells.

In one embodiment, a T cell population is diaglycerol kinase(DGK)-deficient. DGK-deficient cells include cells that do not expressDGK RNA or protein, or have reduced or inhibited DGK activity.DGK-deficient cells can be generated by genetic approaches, e.g.,administering RNA-interfering agents, e.g., siRNA, shRNA, miRNA, toreduce or prevent DGK expression. Alternatively, DGK-deficient cells canbe generated by treatment with DGK inhibitors described herein.

In one embodiment, a T cell population is Ikaros-deficient.Ikaros-deficient cells include cells that do not express Ikaros RNA orprotein, or have reduced or inhibited Ikaros activity, Ikaros-deficientcells can be generated by genetic approaches, e.g., administeringRNA-interfering agents, e.g., siRNA, shRNA, miRNA, to reduce or preventIkaros expression. Alternatively, Ikaros-deficient cells can begenerated by treatment with Ikaros inhibitors, e.g., lenalidomide.

In embodiments, a T cell population is DGK-deficient andIkaros-deficient, e.g., does not express DGK and Ikaros, or has reducedor inhibited DGK and Ikaros activity. Such DGK and Ikaros-deficientcells can be generated by any of the methods described herein.

In an embodiment, the NK cells are obtained from the subject. In anotherembodiment, the NK cells are an NK cell line, e.g., NK-92 cell line(Conkwest).

Additional Expressed Agents

In another embodiment, a CAR-expressing immune effector cell describedherein can further express another agent, e.g., an agent which enhancesthe activity of a CAR-expressing cell. For example, in one embodiment,the agent can be an agent which inhibits an inhibitory molecule.Examples of inhibitory molecules include PD-1, PD-L1, CTLA-4, TIM-3,CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG-3, VISTA, BTLA,TIGIT, LAIR1, CD160, 2B4 and TGFR beta, e.g., as described herein. Inone embodiment, the agent that inhibits an inhibitory molecule comprisesa first polypeptide, e.g., an inhibitory molecule, associated with asecond polypeptide that provides a positive signal to the cell, e.g., anintracellular signaling domain described herein. In one embodiment, theagent comprises a first polypeptide, e.g., of an inhibitory moleculesuch as PD-1, PD-L1, CTLA-4, TIM-3, CEACAM (e.g., CEACAM-1, CEACAM-3and/or CEACAM-5), LAG-3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 or TGFRbeta, or a fragment of any of these, and a second polypeptide which isan intracellular signaling domain described herein (e.g., comprising acostimulatory domain (e.g., 41 BB, CD27 or CD28, e.g., as describedherein) and/or a primary signaling domain (e.g., a CD3 zeta signalingdomain described herein). In one embodiment, the agent comprises a firstpolypeptide of PD-1 or a fragment thereof, and a second polypeptide ofan intracellular signaling domain described herein (e.g., a CD28, CD27,OX40 or 4-IBB signaling domain described herein and/or a CD3 zetasignaling domain described herein).

In one embodiment, the CAR-expressing immune effector cell describedherein can further comprise a second CAR, e.g., a second CAR thatincludes a different antigen binding domain, e.g., to the same target(e.g., a target described above) or a different target. In oneembodiment, the second CAR includes an antigen binding domain to atarget expressed on the same cancer cell type as the target of the firstCAR. In one embodiment, the CAR-expressing immune effector cellcomprises a first CAR that targets a first antigen and includes anintracellular signaling domain having a costimulatory signaling domainbut not a primary signaling domain, and a second CAR that targets asecond, different, antigen and includes an intracellular signalingdomain having a primary signaling domain but not a costimulatorysignaling domain.

While not wishing to be bound by theory, placement of a costimulatorysignaling domain, e.g., 4-1 BB, CD28, CD27 or OX-40, onto the first CAR,and the primary signaling domain, e.g., CD3 zeta, on the second CAR canlimit the CAR activity to cells where both targets are expressed. In oneembodiment, the CAR expressing immune effector cell comprises a firstCAR that includes an antigen binding domain that targets, e.g., a targetdescribed above, a transmembrane domain and a costimulatory domain and asecond CAR that targets an antigen other than antigen targeted by thefirst CAR (e.g., an antigen expressed on the same cancer cell type asthe first target) and includes an antigen binding domain, atransmembrane domain and a primary signaling domain. In anotherembodiment, the CAR expressing immune effector cell comprises a firstCAR that includes an antigen binding domain that targets, e.g., a targetdescribed above, a transmembrane domain and a primary signaling domainand a second CAR that targets an antigen other than antigen targeted bythe first CAR (e.g., an antigen expressed on the same cancer cell typeas the first target) and includes an antigen binding domain to theantigen, a transmembrane domain and a costimulatory signaling domain.

In one embodiment, the CAR-expressing immune effector cell comprises aCAR described herein, e.g., a CAR to a target described above, and aninhibitory CAR. In one embodiment, the inhibitory CAR comprises anantigen binding domain that binds an antigen found on normal cells butnot cancer cells, e.g., normal cells that also express the target. Inone embodiment, the inhibitory CAR comprises the antigen binding domain,a transmembrane domain and an intracellular domain of an inhibitorymolecule. For example, the intracellular domain of the inhibitory CARcan be an intracellular domain of PD1, PD-L1, CTLA-4, TIM-3, CEACAM(e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG-3, VISTA, BTLA, TIGIT,LAIR1, CD160, 2B4 or TGFR beta.

In one embodiment, an immune effector cell (e.g., T cell, NK cell)comprises a first CAR comprising an antigen binding domain that binds toa tumor antigen as described herein, and a second CAR comprising a PD1extracellular domain or a fragment thereof.

In one embodiment, the cell further comprises an inhibitory molecule asdescribed above.

In one embodiment, the second CAR in the cell is an inhibitory CAR,wherein the inhibitory CAR comprises an antigen binding domain, atransmembrane domain, and an intracellular domain of an inhibitorymolecule. The inhibitory molecule can be chosen from one or more of:PD1, PD-L1, CTLA-4, TIM-3, LAG-3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4,TGFR beta, CEACAM-1, CEACAM-3, and CEACAM-5. In one embodiment, thesecond CAR molecule comprises the extracellular domain of PD1 or afragment thereof.

In embodiments, the second CAR molecule in the cell further comprises anintracellular signaling domain comprising a primary signaling domainand/or an intracellular signaling domain.

In other embodiments, the intracellular signaling domain in the cellcomprises a primary signaling domain comprising the functional domain ofCD3 zeta and a costimulatory signaling domain comprising the functionaldomain of 4-1 BB.

In one embodiment, the second CAR molecule in the cell comprises theamino acid sequence of SEQ ID NO: 26.

In certain embodiments, the antigen binding domain of the first CARmolecule comprises a scFv and the antigen binding domain of the secondCAR molecule does not comprise a scFv. For example, the antigen bindingdomain of the first CAR molecule comprises a scFv and the antigenbinding domain of the second CAR molecule comprises a camelid VHHdomain.

Split CAR

In some embodiments, the CAR-expressing cell uses a split CAR. The splitCAR approach is described in more detail in publications WO2014/055442and WO2014/055657. Briefly, a split CAR system comprises a cellexpressing a first CAR having a first antigen binding domain and acostimulatory domain (e.g., 41 BB), and the cell also expresses a secondCAR having a second antigen binding domain and an intracellularsignaling domain (e.g., CD3 zeta). When the cell encounters the firstantigen, the costimulatory domain is activated, and the cellproliferates. When the cell encounters the second antigen, theintracellular signaling domain is activated and cell-killing activitybegins. Thus, the CAR-expressing cell is only fully activated in thepresence of both antigens.

Multiple CAR Expression

In one aspect, the CAR-expressing cell described herein can furthercomprise a second CAR, e.g., a second CAR that includes a differentantigen binding domain, e.g., to the same target or a different target(e.g., a target other than a cancer associated antigen described hereinor a different cancer associated antigen described herein). In oneembodiment, the second CAR includes an antigen binding domain to atarget expressed the same cancer cell type as the cancer associatedantigen. In one embodiment, the CAR-expressing cell comprises a firstCAR that targets a first antigen and includes an intracellular signalingdomain having a costimulatory signaling domain but not a primarysignaling domain, and a second CAR that targets a second, different,antigen and includes an intracellular signaling domain having a primarysignaling domain but not a costimulatory signaling domain. While notwishing to be bound by theory, placement of a costimulatory signalingdomain, e.g., 4-1 BB, CD28, CD27 or OX-40, onto the first CAR, and theprimary signaling domain, e.g., CD3 zeta, on the second CAR can limitthe CAR activity to cells where both targets are expressed. In oneembodiment, the CAR expressing cell comprises a first cancer associatedantigen CAR that includes an antigen binding domain that binds a targetantigen described herein, a transmembrane domain and a costimulatorydomain and a second CAR that targets a different target antigen (e.g.,an antigen expressed on that same cancer cell type as the first targetantigen) and includes an antigen binding domain, a transmembrane domainand a primary signaling domain. In another embodiment, the CARexpressing cell comprises a first CAR that includes an antigen bindingdomain that binds a target antigen described herein, a transmembranedomain and a primary signaling domain and a second CAR that targets anantigen other than the first target antigen (e.g., an antigen expressedon the same cancer cell type as the first target antigen) and includesan antigen binding domain to the antigen, a transmembrane domain and acostimulatory signaling domain.

In some embodiments, the claimed invention comprises a first and secondCAR, wherein the antigen binding domain of one of said first CAR saidsecond CAR does not comprise a variable light domain and a variableheavy domain. In some embodiments, the antigen binding domain of one ofsaid first CAR said second CAR is an scFv, and the other is not an scFv.In some embodiments, the antigen binding domain of one of said first CARsaid second CAR comprises a single VH domain, e.g., a camelid, shark, orlamprey single VH domain, or a single VH domain derived from a human ormouse sequence. In some embodiments, the antigen binding domain of oneof said first CAR said second CAR comprises a nanobody. In someembodiments, the antigen binding domain of one of said first CAR saidsecond CAR comprises a camelid VHH domain.

Telomerase Expression

While not wishing to be bound by any particular theory, in someembodiments, a therapeutic T cell has short term persistence in apatient, due to shortened telomeres in the T cell; accordingly,transfection with a telomerase gene can lengthen the telomeres of the Tcell and improve persistence of the T cell in the patient. See CarlJune, “Adoptive T cell therapy for cancer in the clinic”, Journal ofClinical Investigation, 117:1466-1476 (2007). Thus, in an embodiment, animmune effector cell, e.g., a T cell, ectopically expresses a telomerasesubunit, e.g., the catalytic subunit of telomerase, e.g., TERT, e.g.,hTERT. In some aspects, this disclosure provides a method of producing aCAR-expressing cell, comprising contacting a cell with a nucleic acidencoding a telomerase subunit, e.g., the catalytic subunit oftelomerase, e.g., TERT, e.g., hTERT. The cell may be contacted with thenucleic acid before, simultaneous with, or after being contacted with aconstruct encoding a CAR.

Expansion and Activation

Immune effector cells such as T cells may be activated and expandedgenerally using methods as described, for example, in U.S. Pat. Nos.6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466;6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843;5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent ApplicationPublication No. 20060121005, each of which is incorporated by referencein its entirety.

Generally, a population of immune effector cells e.g., T regulatory celldepleted cells, may be expanded by contact with a surface havingattached thereto an agent that stimulates a CD3/TCR complex associatedsignal and a ligand that stimulates a costimulatory molecule on thesurface of the T cells. In particular, T cell populations may bestimulated as described herein, such as by contact with an anti-CD3antibody, or antigen-binding fragment thereof, or an anti-CD2 antibodyimmobilized on a surface, or by contact with a protein kinase Cactivator (e.g., bryostatin) in conjunction with a calcium ionophore.For co-stimulation of an accessory molecule on the surface of the Tcells, a ligand that binds the accessory molecule is used. For example,a population of T cells can be contacted with an anti-CD3 antibody andan anti-CD28 antibody, under conditions appropriate for stimulatingproliferation of the T cells. To stimulate proliferation of either CD4+T cells or CD8+ T cells, an anti-CD3 antibody and an anti-CD28 antibodycan be used. Examples of an anti-CD28 antibody include 9.3, B-T3,XR-CD28 (Diaclone, Besancon, France) can be used as can other methodscommonly known in the art (Berg et al., Transplant Proc.30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9):13191328,1999; Garland et al., J. Immunol Meth. 227(1-2):53-63, 1999).

In certain aspects, the primary stimulatory signal and the costimulatorysignal for the T cell may be provided by different protocols. Forexample, the agents providing each signal may be in solution or coupledto a surface. When coupled to a surface, the agents may be coupled tothe same surface (i.e., in “cis” formation) or to separate surfaces(i.e., in “trans” formation). Alternatively, one agent may be coupled toa surface and the other agent in solution. In one aspect, the agentproviding the costimulatory signal is bound to a cell surface and theagent providing the primary activation signal is in solution or coupledto a surface. In certain aspects, both agents can be in solution. In oneaspect, the agents may be in soluble form, and then cross-linked to asurface, such as a cell expressing Fc receptors or an antibody or otherbinding agent which will bind to the agents. In this regard, see forexample, U.S. Patent Application Publication Nos. 20040101519 and20060034810 for artificial antigen presenting cells (aAPCs) that arecontemplated for use in activating and expanding T cells in the presentinvention.

In one aspect, the two agents are immobilized on beads, either on thesame bead, i.e., “cis,” or to separate beads, i.e., “trans.” By way ofexample, the agent providing the primary activation signal is ananti-CD3 antibody or an antigen-binding fragment thereof and the agentproviding the costimulatory signal is an anti-CD28 antibody orantigen-binding fragment thereof; and both agents are co-immobilized tothe same bead in equivalent molecular amounts. In one aspect, a 1:1ratio of each antibody bound to the beads for CD4+ T cell expansion andT cell growth is used. In certain aspects of the present invention, aratio of anti CD3:CD28 antibodies bound to the beads is used such thatan increase in T cell expansion is observed as compared to the expansionobserved using a ratio of 1:1. In one particular aspect an increase offrom about 1 to about 3 fold is observed as compared to the expansionobserved using a ratio of 1:1. In one aspect, the ratio of CD3:CD28antibody bound to the beads ranges from 100:1 to 1:100 and all integervalues there between. In one aspect, more anti-CD28 antibody is bound tothe particles than anti-CD3 antibody, i.e., the ratio of CD3:CD28 isless than one. In certain aspects, the ratio of anti CD28 antibody toanti CD3 antibody bound to the beads is greater than 2:1. In oneparticular aspect, a 1:100 CD3:CD28 ratio of antibody bound to beads isused. In one aspect, a 1:75 CD3:CD28 ratio of antibody bound to beads isused. In a further aspect, a 1:50 CD3:CD28 ratio of antibody bound tobeads is used. In one aspect, a 1:30 CD3:CD28 ratio of antibody bound tobeads is used. In one preferred aspect, a 1:10 CD3:CD28 ratio ofantibody bound to beads is used. In one aspect, a 1:3 CD3:CD28 ratio ofantibody bound to the beads is used. In yet one aspect, a 3:1 CD3:CD28ratio of antibody bound to the beads is used.

Ratios of particles to cells from 1:500 to 500:1 and any integer valuesin between may be used to stimulate T cells or other target cells. Asthose of ordinary skill in the art can readily appreciate, the ratio ofparticles to cells may depend on particle size relative to the targetcell. For example, small sized beads could only bind a few cells, whilelarger beads could bind many. In certain aspects the ratio of cells toparticles ranges from 1:100 to 100:1 and any integer values in-betweenand in further aspects the ratio comprises 1:9 to 9:1 and any integervalues in between, can also be used to stimulate T cells. The ratio ofanti-CD3- and anti-CD28-coupled particles to T cells that result in Tcell stimulation can vary as noted above, however certain preferredvalues include 1:100, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8, 1:7, 1:6,1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1,and 15:1 with one preferred ratio being at least 1:1 particles per Tcell. In one aspect, a ratio of particles to cells of 1:1 or less isused. In one particular aspect, a preferred particle: cell ratio is 1:5.In further aspects, the ratio of particles to cells can be varieddepending on the day of stimulation. For example, in one aspect, theratio of particles to cells is from 1:1 to 10:1 on the first day andadditional particles are added to the cells every day or every other daythereafter for up to 10 days, at final ratios of from 1:1 to 1:10 (basedon cell counts on the day of addition). In one particular aspect, theratio of particles to cells is 1:1 on the first day of stimulation andadjusted to 1:5 on the third and fifth days of stimulation. In oneaspect, particles are added on a daily or every other day basis to afinal ratio of 1:1 on the first day, and 1:5 on the third and fifth daysof stimulation. In one aspect, the ratio of particles to cells is 2:1 onthe first day of stimulation and adjusted to 1:10 on the third and fifthdays of stimulation. In one aspect, particles are added on a daily orevery other day basis to a final ratio of 1:1 on the first day, and 1:10on the third and fifth days of stimulation. One of skill in the art willappreciate that a variety of other ratios may be suitable for use in thepresent invention. In particular, ratios will vary depending on particlesize and on cell size and type. In one aspect, the most typical ratiosfor use are in the neighborhood of 1:1, 2:1 and 3:1 on the first day.

In further aspects, the cells, such as T cells, are combined withagent-coated beads, the beads and the cells are subsequently separated,and then the cells are cultured. In an alternative aspect, prior toculture, the agent-coated beads and cells are not separated but arecultured together. In a further aspect, the beads and cells are firstconcentrated by application of a force, such as a magnetic force,resulting in increased ligation of cell surface markers, therebyinducing cell stimulation.

By way of example, cell surface proteins may be ligated by allowingparamagnetic beads to which anti-CD3 and anti-CD28 are attached (3×28beads) to contact the T cells. In one aspect the cells (for example, 10⁴to 10⁹T cells) and beads (for example, DYNABEADS® M-450 CD3/CD28 Tparamagnetic beads at a ratio of 1:1) are combined in a buffer, forexample PBS (without divalent cations such as, calcium and magnesium).Again, those of ordinary skill in the art can readily appreciate anycell concentration may be used. For example, the target cell may be veryrare in the sample and comprise only 0.01% of the sample or the entiresample (i.e., 100%) may comprise the target cell of interest.Accordingly, any cell number is within the context of the presentinvention. In certain aspects, it may be desirable to significantlydecrease the volume in which particles and cells are mixed together(i.e., increase the concentration of cells), to ensure maximum contactof cells and particles. For example, in one aspect, a concentration ofabout 10 billion cells/ml, 9 billion/ml, 8 billion/ml, 7 billion/ml, 6billion/ml, 5 billion/ml, or 2 billion cells/ml is used. In one aspect,greater than 100 million cells/ml is used. In a further aspect, aconcentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 millioncells/ml is used. In yet one aspect, a concentration of cells from 75,80, 85, 90, 95, or 100 million cells/ml is used. In further aspects,concentrations of 125 or 150 million cells/ml can be used. Using highconcentrations can result in increased cell yield, cell activation, andcell expansion. Further, use of high cell concentrations allows moreefficient capture of cells that may weakly express target antigens ofinterest, such as CD28-negative T cells. Such populations of cells mayhave therapeutic value and would be desirable to obtain in certainaspects. For example, using high concentration of cells allows moreefficient selection of CD8+ T cells that normally have weaker CD28expression.

In one embodiment, cells transduced with a nucleic acid encoding a CAR,e.g., a CAR described herein, are expanded, e.g., by a method describedherein. In one embodiment, the cells are expanded in culture for aperiod of several hours (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 18,21 hours) to about 14 days (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13 or 14 days). In one embodiment, the cells are expanded for a periodof 4 to 9 days. In one embodiment, the cells are expanded for a periodof 8 days or less, e.g., 7, 6 or 5 days. In one embodiment, the cellsare expanded in culture for 5 days, and the resulting cells are morepotent than the same cells expanded in culture for 9 days under the sameculture conditions. Potency can be defined, e.g., by various T cellfunctions, e.g. proliferation, target cell killing, cytokine production,activation, migration, or combinations thereof. In one embodiment, thecells are expanded for 5 days show at least a one, two, three or fourfold increase in cells doublings upon antigen stimulation as compared tothe same cells expanded in culture for 9 days under the same cultureconditions. In one embodiment, the cells are expanded in culture for 5days, and the resulting cells exhibit higher proinflammatory cytokineproduction, e.g., IFN-γ and/or GM-CSF levels, as compared to the samecells expanded in culture for 9 days under the same culture conditions.In one embodiment, the cells expanded for 5 days show at least a one,two, three, four, five, ten fold or more increase in pg/ml ofproinflammatory cytokine production, e.g., IFN-γ and/or GM-CSF levels,as compared to the same cells expanded in culture for 9 days under thesame culture conditions.

Several cycles of stimulation may also be desired such that culture timeof T cells can be 60 days or more. Conditions appropriate for T cellculture include an appropriate media (e.g., Minimal Essential Media orRPMI Media 1640 or, X-vivo 15, (Lonza)) that may contain factorsnecessary for proliferation and viability, including serum (e.g., fetalbovine or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4,IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFβ, and TNF-α or any otheradditives for the growth of cells known to the skilled artisan. Otheradditives for the growth of cells include, but are not limited to,surfactant, plasmanate, and reducing agents such as N-acetyl-cysteineand 2-mercaptoethanol. Media can include RPMI 1640, AIM-V, DMEM, MEM,α-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, with added aminoacids, sodium pyruvate, and vitamins, either serum-free or supplementedwith an appropriate amount of serum (or plasma) or a defined set ofhormones, and/or an amount of cytokine(s) sufficient for the growth andexpansion of T cells. Antibiotics, e.g., penicillin and streptomycin,are included only in experimental cultures, not in cultures of cellsthat are to be infused into a subject. The target cells are maintainedunder conditions necessary to support growth, for example, anappropriate temperature (e.g., 37° C.) and atmosphere (e.g., air plus 5%CO₂).

In one embodiment, the cells are expanded in an appropriate media (e.g.,media described herein) that includes one or more interleukin thatresult in at least a 200-fold (e.g., 200-fold, 250-fold, 300-fold,350-fold) increase in cells over a 14 day expansion period, e.g., asmeasured by a method described herein such as flow cytometry. In oneembodiment, the cells are expanded in the presence of IL-15 and/or IL-7(e.g., IL-15 and IL-7).

In embodiments, methods described herein, e.g., CAR-expressing cellmanufacturing methods, comprise removing T regulatory cells, e.g., CD25+T cells, from a cell population, e.g., using an anti-CD25 antibody, orfragment thereof, or a CD25-binding ligand, IL-2. Methods of removing Tregulatory cells, e.g., CD25+ T cells, from a cell population aredescribed herein. In embodiments, the methods, e.g., manufacturingmethods, further comprise contacting a cell population (e.g., a cellpopulation in which T regulatory cells, such as CD25+ T cells, have beendepleted; or a cell population that has previously contacted ananti-CD25 antibody, fragment thereof, or CD25-binding ligand) with IL-15and/or IL-7. For example, the cell population (e.g., that has previouslycontacted an anti-CD25 antibody, fragment thereof, or CD25-bindingligand) is expanded in the presence of IL-15 and/or IL-7.

In some embodiments a CAR-expressing cell described herein is contactedwith a composition comprising a interleukin-15 (IL-15) polypeptide, ainterleukin-15 receptor alpha (IL-15Ra) polypeptide, or a combination ofboth a IL-15 polypeptide and a IL-15Ra polypeptide e.g., hetIL-15,during the manufacturing of the CAR-expressing cell, e.g., ex vivo. Inembodiments, a CAR-expressing cell described herein is contacted with acomposition comprising a IL-15 polypeptide during the manufacturing ofthe CAR-expressing cell, e.g., ex vivo. In embodiments, a CAR-expressingcell described herein is contacted with a composition comprising acombination of both a IL-15 polypeptide and a IL-15 Ra polypeptideduring the manufacturing of the CAR-expressing cell, e.g., ex vivo. Inembodiments, a CAR-expressing cell described herein is contacted with acomposition comprising hetIL-15 during the manufacturing of theCAR-expressing cell, e.g., ex vivo.

In one embodiment the CAR-expressing cell described herein is contactedwith a composition comprising hetIL-15 during ex vivo expansion. In anembodiment, the CAR-expressing cell described herein is contacted with acomposition comprising an IL-15 polypeptide during ex vivo expansion. Inan embodiment, the CAR-expressing cell described herein is contactedwith a composition comprising both an IL-15 polypeptide and an IL-15Rapolypeptide during ex vivo expansion. In one embodiment the contactingresults in the survival and proliferation of a lymphocyte subpopulation,e.g., CD8+ T cells.

T cells that have been exposed to varied stimulation times may exhibitdifferent characteristics. For example, typical blood or apheresedperipheral blood mononuclear cell products have a helper T cellpopulation (TH, CD4+) that is greater than the cytotoxic or suppressor Tcell population (TC, CD8+). Ex vivo expansion of T cells by stimulatingCD3 and CD28 receptors produces a population of T cells that prior toabout days 8-9 consists predominately of TH cells, while after aboutdays 8-9, the population of T cells comprises an increasingly greaterpopulation of TC cells. Accordingly, depending on the purpose oftreatment, infusing a subject with a T cell population comprisingpredominately of TH cells may be advantageous. Similarly, if anantigen-specific subset of TC cells has been isolated it may bebeneficial to expand this subset to a greater degree.

Further, in addition to CD4 and CD8 markers, other phenotypic markersvary significantly, but in large part, reproducibly during the course ofthe cell expansion process. Thus, such reproducibility enables theability to tailor an activated T cell product for specific purposes.

Once a CAR described herein is constructed, various assays can be usedto evaluate the activity of the molecule, such as but not limited to,the ability to expand T cells following antigen stimulation, sustain Tcell expansion in the absence of re-stimulation, and anti-canceractivities in appropriate in vitro and animal models. Assays to evaluatethe effects of a cars of the present invention are described in furtherdetail below

Western blot analysis of CAR expression in primary T cells can be usedto detect the presence of monomers and dimers. See, e.g., Milone et al.,Molecular Therapy 17(8): 1453-1464 (2009). Very briefly, T cells (1:1mixture of CD4+ and CD8+ T cells) expressing the CARs are expanded invitro for more than 10 days followed by lysis and SDS-PAGE underreducing conditions. CARs containing the full length TCR-ζ cytoplasmicdomain and the endogenous TCR-ζ chain are detected by western blottingusing an antibody to the TCR-ζ chain. The same T cell subsets are usedfor SDS-PAGE analysis under non-reducing conditions to permit evaluationof covalent dimer formation.

In vitro expansion of CAR⁺ T cells following antigen stimulation can bemeasured by flow cytometry. For example, a mixture of CD4⁺ and CD8⁺ Tcells are stimulated with αCD3/αCD28 aAPCs followed by transduction withlentiviral vectors expressing GFP under the control of the promoters tobe analyzed. Exemplary promoters include the CMV IE gene, EF-1α,ubiquitin C, or phosphoglycerokinase (PGK) promoters. GFP fluorescenceis evaluated on day 6 of culture in the CD4⁺ and/or CD8⁺ T cell subsetsby flow cytometry. See, e.g., Milone et al., Molecular Therapy 17(8):1453-1464 (2009). Alternatively, a mixture of CD4⁺ and CD8⁺ T cells arestimulated with αCD3/αCD28 coated magnetic beads on day 0, andtransduced with CAR on day 1 using a bicistronic lentiviral vectorexpressing CAR along with eGFP using a 2A ribosomal skipping sequence.Cultures are re-stimulated with either a cancer associated antigen asdescribed herein⁺ K562 cells (K562 expressing a cancer associatedantigen as described herein), wild-type K562 cells (K562 wild type) orK562 cells expressing hCD32 and 4-1 BBL in the presence of antiCD3 andanti-CD28 antibody (K562-BBL-3/28) following washing. Exogenous IL-2 isadded to the cultures every other day at 100 IU/ml. GFP⁺ T cells areenumerated by flow cytometry using bead-based counting. See, e.g.,Milone et al., Molecular Therapy 17(8): 1453-1464 (2009).

Sustained CAR⁺ T cell expansion in the absence of re-stimulation canalso be measured. See, e.g., Milone et al., Molecular Therapy 17(8):1453-1464 (2009). Briefly, mean T cell volume (fl) is measured on day 8of culture using a Coulter Multisizer III particle counter, a NexcelomCellometer Vision or Millipore Scepter, following stimulation withαCD3/αCD28 coated magnetic beads on day 0, and transduction with theindicated CAR on day 1.

Animal models can also be used to measure a CART activity. For example,xenograft model using human a cancer associated antigen describedherein-specific CAR⁺ T cells to treat a primary human pre-B ALL inimmunodeficient mice can be used. See, e.g., Milone et al., MolecularTherapy 17(8): 1453-1464 (2009). Very briefly, after establishment ofALL, mice are randomized as to treatment groups. Different numbers of acancer associated antigen-specific CARengineered T cells are coinjectedat a 1:1 ratio into NOD-SCID-γ^(−/−) mice bearing B-ALL. The number ofcopies of a cancer associated antigen-specific CAR vector in spleen DNAfrom mice is evaluated at various times following T cell injection.Animals are assessed for leukemia at weekly intervals. Peripheral blooda cancer associate antigen as described herein⁺ B-ALL blast cell countsare measured in mice that are injected with a cancer associated antigendescribed herein-ζ CAR⁺ T cells or mock-transduced T cells. Survivalcurves for the groups are compared using the log-rank test. In addition,absolute peripheral blood CD4⁺ and CD8⁺ T cell counts 4 weeks followingT cell injection in NOD-SCID-γ^(−/−) mice can also be analyzed. Mice areinjected with leukemic cells and 3 weeks later are injected with T cellsengineered to express CAR by a bicistronic lentiviral vector thatencodes the CAR linked to eGFP. T cells are normalized to 45-50% inputGFP⁺ T cells by mixing with mock-transduced cells prior to injection,and confirmed by flow cytometry. Animals are assessed for leukemia at1-week intervals. Survival curves for the CAR⁺ T cell groups arecompared using the log-rank test.

Dose dependent CAR treatment response can be evaluated. See, e.g.,Milone et al., Molecular Therapy 17(8): 1453-1464 (2009). For example,peripheral blood is obtained 35-70 days after establishing leukemia inmice injected on day 21 with CAR T cells, an equivalent number ofmock-transduced T cells, or no T cells. Mice from each group arerandomly bled for determination of peripheral blood a cancer associateantigen as described herein⁺ ALL blast counts and then killed on days 35and 49. The remaining animals are evaluated on days 57 and 70.

Assessment of cell proliferation and cytokine production has beenpreviously described, e.g., at Milone et al., Molecular Therapy 17(8):1453-1464 (2009). Briefly, assessment of CAR-mediated proliferation isperformed in microtiter plates by mixing washed T cells with K562 cellsexpressing a cancer associated antigen described herein (K19) or CD32and CD137 (KT32-BBL) for a final T-cell:K562 ratio of 2:1. K562 cellsare irradiated with gamma-radiation prior to use. Anti-CD3 (clone OKT3)and anti-CD28 (clone 9.3) monoclonal antibodies are added to cultureswith KT32-BBL cells to serve as a positive control for stimulatingT-cell proliferation since these signals support long-term CD8⁺ T cellexpansion ex vivo. T cells are enumerated in cultures using CountBright™fluorescent beads (Invitrogen, Carlsbad, Calif.) and flow cytometry asdescribed by the manufacturer. CAR⁺ T cells are identified by GFPexpression using T cells that are engineered with eGFP-2A linkedCAR-expressing lentiviral vectors. For CAR+ T cells not expressing GFP,the CAR+ T cells are detected with biotinylated recombinant a cancerassociate antigen as described herein protein and a secondary avidin-PEconjugate. CD4+ and CD8⁺ expression on T cells are also simultaneouslydetected with specific monoclonal antibodies (BD Biosciences). Cytokinemeasurements are performed on supernatants collected 24 hours followingre-stimulation using the human TH1/TH2 cytokine cytometric bead arraykit (BD Biosciences, San Diego, Calif.) according the manufacturer'sinstructions. Fluorescence is assessed using a FACScalibur flowcytometer, and data is analyzed according to the manufacturer'sinstructions.

Cytotoxicity can be assessed by a standard 51Cr-release assay. See,e.g., Milone et al., Molecular Therapy 17(8): 1453-1464 (2009). Briefly,target cells (K562 lines and primary pro-B-ALL cells) are loaded with51Cr (as NaCrO4, New England Nuclear, Boston, Mass.) at 37° C. for 2hours with frequent agitation, washed twice in complete RPMI and platedinto microtiter plates. Effector T cells are mixed with target cells inthe wells in complete RPMI at varying ratios of effector cell:targetcell (E:T). Additional wells containing media only (spontaneous release,SR) or a 1% solution of triton-X 100 detergent (total release, TR) arealso prepared. After 4 hours of incubation at 37° C., supernatant fromeach well is harvested. Released 51Cr is then measured using a gammaparticle counter (Packard Instrument Co., Waltham, Mass.). Eachcondition is performed in at least triplicate, and the percentage oflysis is calculated using the formula: % Lysis=(ER−SR)/(TR−SR), where ERrepresents the average 51Cr released for each experimental condition.

Imaging technologies can be used to evaluate specific trafficking andproliferation of CARs in tumor-bearing animal models. Such assays havebeen described, for example, in Barrett et al., Human Gene Therapy22:1575-1586 (2011). Briefly, NOD/SCID/γc^(−/−) (NSG) mice are injectedIV with Nalm-6 cells followed 7 days later with T cells 4 hour afterelectroporation with the CAR constructs. The T cells are stablytransfected with a lentiviral construct to express firefly luciferase,and mice are imaged for bioluminescence. Alternatively, therapeuticefficacy and specificity of a single injection of CAR⁺ T cells in Nalm-6xenograft model can be measured as the following: NSG mice are injectedwith Nalm-6 transduced to stably express firefly luciferase, followed bya single tail-vein injection of T cells electroporated with cars of thepresent invention 7 days later. Animals are imaged at various timepoints post injection. For example, photon-density heat maps of fireflyluciferasepositive leukemia in representative mice at day 5 (2 daysbefore treatment) and day 8 (24 hr post CAR⁺ PBLs) can be generated.

Other assays, including those described in the Example section herein aswell as those that are known in the art can also be used to evaluate theCARs described herein.

Methods of Treatment/Combination Therapies

In another aspect, the present invention provides a method comprisingadministering a CAR molecule, e.g., a CAR molecule described herein, ora cell comprising a nucleic acid encoding a CAR molecule, e.g., a CARmolecule described herein. In one embodiment, the subject has a disorderdescribed herein, e.g., the subject has cancer, e.g., the subject has acancer which expresses a target antigen described herein. In oneembodiment, the subject is a human.

In another aspect, the invention pertains to a method of treating asubject having a disease associated with expression of a cancerassociated antigen as described herein comprising administering to thesubject an effective amount of a cell comprising a CAR molecule, e.g., aCAR molecule described herein.

In yet another aspect, the invention features a method of treating asubject having a disease associated with expression of a tumor antigen(e.g., an antigen described herein), comprising administering to thesubject an effective amount of a cell, e.g., an immune effector cell(e.g., a population of immune effector cells) comprising a CAR molecule,wherein the CAR molecule comprises an antigen binding domain, atransmembrane domain, and an intracellular domain, said intracellulardomain comprises a costimulatory domain and/or a primary signalingdomain, wherein said antigen binding domain binds to the tumor antigenassociated with the disease, e.g. a tumor antigen as described herein.

In a related aspect, the invention features a method of treating asubject having a disease associated with expression of a tumor antigen.The method comprises administering to the subject an effective amount ofa cell, e.g., an immune effector cell (e.g., a population of immuneeffector cells) comprising a CAR molecule, in combination with an agentthat increases the efficacy of the immune cell, wherein:

the agent that increases the efficacy of the immune cell is chosen fromone or more of:

(i) a protein phosphatase inhibitor;

(ii) a kinase inhibitor;

(iii) a cytokine;

(iv) an inhibitor of an immune inhibitory molecule; or

(v) an agent that decreases the level or activity of a T_(REG) cell.

In another aspect, the invention features a composition comprising animmune effector cell (e.g., a population of immune effector cells)comprising a CAR molecule (e.g., a CAR molecule as described herein) foruse in the treatment of a subject having a disease associated withexpression of a tumor antigen, e.g., a disorder as described herein.

In certain embodiments of any of the aforesaid methods or uses, thedisease associated with a tumor antigen, e.g., a tumor antigen describedherein, is selected from a proliferative disease such as a cancer ormalignancy or a precancerous condition such as a myelodysplasia, amyelodysplastic syndrome or a preleukemia, or is a non-cancer relatedindication associated with expression of a tumor antigen describedherein. In one embodiment, the disease is a cancer described herein,e.g., a cancer described herein as being associated with a targetdescribed herein. In one embodiment, the disease is a hematologiccancer. In one embodiment, the hematologic cancer is leukemia. In oneembodiment, the cancer is selected from the group consisting of one ormore acute leukemias including but not limited to B-cell acute lymphoidleukemia (“BALL”), T-cell acute lymphoid leukemia (“TALL”), acutelymphoid leukemia (ALL); one or more chronic leukemias including but notlimited to chronic myelogenous leukemia (CML), chronic lymphocyticleukemia (CLL); additional hematologic cancers or hematologic conditionsincluding, but not limited to B cell prolymphocytic leukemia, blasticplasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse largeB cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell-or a large cell-follicular lymphoma, malignant lymphoproliferativeconditions, MALT lymphoma, mantle cell lymphoma, Marginal zone lymphoma,multiple myeloma, myelodysplasia and myelodysplastic syndrome,non-Hodgkin lymphoma, Hodgkin lymphoma, plasmablastic lymphoma,plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia, and“preleukemia” which are a diverse collection of hematological conditionsunited by ineffective production (or dysplasia) of myeloid blood cells,and to disease associated with expression of a tumor antigen describedherein include, but not limited to, atypical and/or non-classicalcancers, malignancies, precancerous conditions or proliferative diseasesexpressing a tumor antigen as described herein; and any combinationthereof. In another embodiment, the disease associated with a tumorantigen described herein is a solid tumor.

In certain embodiments, the methods or uses are carried out incombination with an agent that increases the efficacy of the immuneeffector cell, e.g., an agent as described herein.

In any of the aforesaid methods or uses, the disease associated withexpression of the tumor antigen is selected from the group consisting ofa proliferative disease, a precancerous condition, a cancer, and anon-cancer related indication associated with expression of the tumorantigen.

The cancer can be a hematologic cancer, e.g., a cancer chosen from oneor more of chronic lymphocytic leukemia (CLL), acute leukemias, acutelymphoid leukemia (ALL), B-cell acute lymphoid leukemia (B-ALL), T-cellacute lymphoid leukemia (T-ALL), chronic myelogenous leukemia (CML), Bcell prolymphocytic leukemia, blastic plasmacytoid dendritic cellneoplasm, Burkitt's lymphoma, diffuse large B cell lymphoma, follicularlymphoma, hairy cell leukemia, small cell- or a large cell-follicularlymphoma, malignant lymphoproliferative conditions, MALT lymphoma,mantle cell lymphoma, marginal zone lymphoma, multiple myeloma,myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma,Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cellneoplasm, Waldenstrom macroglobulinemia, or pre-leukemia.

The cancer can also be chosen from colon cancer, rectal cancer,renal-cell carcinoma, liver cancer, non-small cell carcinoma of thelung, cancer of the small intestine, cancer of the esophagus, melanoma,bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck,cutaneous or intraocular malignant melanoma, uterine cancer, ovariancancer, rectal cancer, cancer of the anal region, stomach cancer,testicular cancer, uterine cancer, carcinoma of the fallopian tubes,carcinoma of the endometrium, carcinoma of the cervix, carcinoma of thevagina, carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin'slymphoma, cancer of the endocrine system, cancer of the thyroid gland,cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma ofsoft tissue, cancer of the urethra, cancer of the penis, solid tumors ofchildhood, cancer of the bladder, cancer of the kidney or ureter,carcinoma of the renal pelvis, neoplasm of the central nervous system(CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor,brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoidcancer, squamous cell cancer, T-cell lymphoma, environmentally inducedcancers, combinations of said cancers, and metastatic lesions of saidcancers.

In certain embodiments of the methods or uses described herein, the CARmolecule is administered in combination with an agent that increases theefficacy of the immune effector cell, e.g., one or more of a proteinphosphatase inhibitor, a kinase inhibitor, a cytokine, an inhibitor ofan immune inhibitory molecule; or an agent that decreases the level oractivity of a T_(REG) cell.

In certain embodiments of the methods or uses described herein, theprotein phosphatase inhibitor is a SHP-1 inhibitor and/or an SHP-2inhibitor.

In other embodiments of the methods or uses described herein, kinaseinhibitor is chosen from one or more of a CDK4 inhibitor, a CDK4/6inhibitor (e.g., palbociclib), a BTK inhibitor (e.g., ibrutinib orRN-486), an mTOR inhibitor (e.g., rapamycin or everolimus (RAD001)), anMNK inhibitor, or a dual PI3K/mTOR inhibitor. In one embodiment, the BTKinhibitor does not reduce or inhibit the kinase activity ofinterleukin-2-inducible kinase (ITK).

In other embodiments of the methods or uses described herein, the agentthat inhibits the immune inhibitory molecule comprises an antibody orantibody fragment, an inhibitory nucleic acid, a clustered regularlyinterspaced short palindromic repeats (CRISPR), atranscription-activator like effector nuclease (TALEN), or a zinc fingerendonuclease (ZFN) that inhibits the expression of the inhibitorymolecule.

In other embodiments of the methods or uses described herein, the agentthat decreases the level or activity of the T_(REG) cells is chosen fromcyclophosphamide, anti-GITR antibody, CD25-depletion, or a combinationthereof.

In certain embodiments of the methods or uses described herein, theimmune inhibitory molecule is selected from the group consisting of PD1,PD-L1, CTLA-4, TIM-3, LAG-3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, TGFRbeta, CEACAM-1, CEACAM-3, and CEACAM-5.

In other embodiments, the agent that inhibits the inhibitory moleculecomprises a first polypeptide comprising an inhibitory molecule or afragment thereof and a second polypeptide that provides a positivesignal to the cell, and wherein the first and second polypeptides areexpressed on the CAR-containing immune cells, wherein (i) the firstpolypeptide comprises PD1, PD-L1, CTLA-4, TIM-3, LAG3, VISTA, BTLA,TIGIT, LAIR1, CD160, 2B4, TGFR beta, CEACAM-1, CEACAM-3, and CEACAM-5 ora fragment thereof; and/or (ii) the second polypeptide comprises anintracellular signaling domain comprising a primary signaling domainand/or a costimulatory signaling domain. In one embodiment, the primarysignaling domain comprises a functional domain of CD3 zeta; and/or thecostimulatory signaling domain comprises a functional domain of aprotein selected from 41 BB, CD27 and CD28.

In other embodiments, cytokine is chosen from IL-7, IL-15 or IL-21, orboth.

In other embodiments, the immune effector cell comprising the CARmolecule and a second, e.g., any of the combination therapies disclosedherein (e.g., the agent that that increases the efficacy of the immuneeffector cell) are administered substantially simultaneously orsequentially.

In other embodiments, the immune cell comprising the CAR molecule isadministered in combination with a molecule that targets GITR and/ormodulates GITR function. In certain embodiments, the molecule targetingGITR and/or modulating GITR function is administered prior to theCAR-expressing cell or population of cells, or prior to apheresis.

In one embodiment, lymphocyte infusion, for example allogeneiclymphocyte infusion, is used in the treatment of the cancer, wherein thelymphocyte infusion comprises at least one CAR-expressing cell of thepresent invention. In one embodiment, autologous lymphocyte infusion isused in the treatment of the cancer, wherein the autologous lymphocyteinfusion comprises at least one CAR-expressing cell described herein.

In one embodiment, the cell is a T cell and the T cell is diaglycerolkinase (DGK) deficient. In one embodiment, the cell is a T cell and theT cell is Ikaros deficient. In one embodiment, the cell is a T cell andthe T cell is both DGK and Ikaros deficient.

In one embodiment, the method includes administering a cell expressingthe CAR molecule, as described herein, in combination with an agentwhich enhances the activity of a CAR-expressing cell, wherein the agentis a cytokine, e.g., IL-7, IL-15, IL-18, IL-21, or a combinationthereof. The cytokine can be delivered in combination with, e.g.,simultaneously or shortly after, administration of the CAR-expressingcell. Alternatively, the cytokine can be delivered after a prolongedperiod of time after administration of the CAR-expressing cell, e.g.,after assessment of the subject's response to the CAR-expressing cell.In one embodiment the cytokine is administered to the subjectsimultaneously (e.g., administered on the same day) with or shortlyafter administration (e.g., administered 1 day, 2 days, 3 days, 4 days,5 days, 6 days, or 7 days after administration) of the cell orpopulation of cells of any of claims 61-80. In other embodiments, thecytokine is administered to the subject after a prolonged period of time(e.g., e.g., at least 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 10weeks, or more) after administration of the cell or population of cellsof any of claims 61-80, or after assessment of the subject's response tothe cell.

In other embodiments, the cells expressing a CAR molecule areadministered in combination with an agent that ameliorates one or moreside effects associated with administration of a cell expressing a CARmolecule. Side effects associated with the CAR-expressing cell can bechosen from cytokine release syndrome (CRS) or hemophagocyticlymphohistiocytosis (HLH).

In embodiments of any of the aforeseaid methods or uses, the cellsexpressing the CAR molecule are administered in combination with anagent that treats the disease associated with expression of the tumorantigen, e.g., any of the second or third therapies disclosed herein.Additional exemplary combinations include one or more of the following.

In another embodiment, the cell expressing the CAR molecule, e.g., asdescribed herein, can be administered in combination with another agent,e.g., a kinase inhibitor and/or checkpoint inhibitor described herein.In an embodiment, a cell expressing the CAR molecule can further expressanother agent, e.g., an agent which enhances the activity of aCAR-expressing cell.

For example, in one embodiment, the agent that enhances the activity ofa CAR-expressing cell can be an agent which inhibits an inhibitorymolecule (e.g., an immune inhibitor molecule). Examples of inhibitorymolecules include PD1, PD-L1, CTLA-4, TIM-3, CEACAM (e.g., CEACAM-1,CEACAM-3 and/or CEACAM-5), LAG-3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4and TGFR beta.

In one embodiment, the agent that inhibits the inhibitory molecule is aninhibitory nucleic acid is a dsRNA, a siRNA, or a shRNA. In embodiments,the inhibitory nucleic acid is linked to the nucleic acid that encodes acomponent of the CAR molecule. For example, the inhibitory molecule canbe expressed on the CAR-expressing cell.

In another embodiment, the agent which inhibits an inhibitory molecule,e.g., is a molecule described herein, e.g., an agent that comprises afirst polypeptide, e.g., an inhibitory molecule, associated with asecond polypeptide that provides a positive signal to the cell, e.g., anintracellular signaling domain described herein. In one embodiment, theagent comprises a first polypeptide, e.g., of an inhibitory moleculesuch as PD-1, PD-L1, CTLA-4, TIM-3, CEACAM (e.g., CEACAM-1, CEACAM-3and/or CEACAM-5), LAG-3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 or TGFRbeta, or a fragment of any of these (e.g., at least a portion of theextracellular domain of any of these), and a second polypeptide which isan intracellular signaling domain described herein (e.g., comprising acostimulatory domain (e.g., 41 BB, CD27 or CD28, e.g., as describedherein) and/or a primary signaling domain (e.g., a CD3 zeta signalingdomain described herein). In one embodiment, the agent comprises a firstpolypeptide of PD1 or a fragment thereof (e.g., at least a portion ofthe extracellular domain of PD1), and a second polypeptide of anintracellular signaling domain described herein (e.g., a CD28 signalingdomain described herein and/or a CD3 zeta signaling domain describedherein).

In one embodiment, the CAR-expressing immune effector cell of thepresent invention, e.g., T cell or NK cell, is administered to a subjectthat has received a previous stem cell transplantation, e.g., autologousstem cell transplantation.

In one embodiment, the CAR-expressing immune effector cell of thepresent invention, e.g., T cell or NK cells, is administered to asubject that has received a previous dose of melphalan.

In one embodiment, the cell expressing a CAR molecule, e.g., a CARmolecule described herein, is administered in combination with an agentthat increases the efficacy of a cell expressing a CAR molecule, e.g.,an agent described herein.

In one embodiment, the cells expressing a CAR molecule, e.g., a CARmolecule described herein, are administered in combination with a low,immune enhancing dose of an mTOR inhibitor. While not wishing to bebound by theory, it is believed that treatment with a low, immuneenhancing, dose (e.g., a dose that is insufficient to completelysuppress the immune system but sufficient to improve immune function) isaccompanied by a decrease in PD-1 positive T cells or an increase inPD-1 negative cells. PD-1 positive T cells, but not PD-1 negative Tcells, can be exhausted by engagement with cells which express a PD-1ligand, e.g., PD-L1 or PD-L2.

In an embodiment this approach can be used to optimize the performanceof CAR cells described herein in the subject. While not wishing to bebound by theory, it is believed that, in an embodiment, the performanceof endogenous, non-modified immune effector cells, e.g., T cells or NKcells, is improved. While not wishing to be bound by theory, it isbelieved that, in an embodiment, the performance of a target antigenCAR-expressing cell is improved. In other embodiments, cells, e.g., Tcells or NK cells, which have, or will be engineered to express a CAR,can be treated ex vivo by contact with an amount of an mTOR inhibitorthat increases the number of PD1 negative immune effector cells, e.g., Tcells or increases the ratio of PD1 negative immune effector cells,e.g., T cells/PD1 positive immune effector cells, e.g., T cells.

In an embodiment, administration of a low, immune enhancing, dose of anmTOR inhibitor, e.g., an allosteric inhibitor, e.g., RAD001, or acatalytic inhibitor, is initiated prior to administration of an CARexpressing cell described herein, e.g., T cells or NK cells. In anembodiment, the CAR cells are administered after a sufficient time, orsufficient dosing, of an mTOR inhibitor, such that the level of PD1negative immune effector cells, e.g., T cells or NK cells, or the ratioof PD1 negative immune effector cells, e.g., T cells/PD1 positive immuneeffector cells, e.g., T cells, has been, at least transiently,increased.

In an embodiment, the cell, e.g., T cell or NK cell, to be engineered toexpress a CAR, is harvested after a sufficient time, or after sufficientdosing of the low, immune enhancing, dose of an mTOR inhibitor, suchthat the level of PD1 negative immune effector cells, e.g., T cells, orthe ratio of PD1 negative immune effector cells, e.g., T cells/PD1positive immune effector cells, e.g., T cells, in the subject orharvested from the subject has been, at least transiently, increased.

In one embodiment, the cell expressing a CAR molecule, e.g., a CARmolecule described herein, is administered in combination with an agentthat ameliorates one or more side effect associated with administrationof a cell expressing a CAR molecule, e.g., an agent described herein.

In one embodiment, the cell expressing a CAR molecule, e.g., a CARmolecule described herein, is administered in combination with an agentthat treats the disease associated with a cancer associated antigen asdescribed herein, e.g., an agent described herein.

In one embodiment, a cell expressing two or more CAR molecules, e.g., asdescribed herein, is administered to a subject in need thereof to treatcancer. In one embodiment, a population of cells including a CARexpressing cell, e.g., as described herein, is administered to a subjectin need thereof to treat cancer.

In one embodiment, the cell expressing a CAR molecule, e.g., a CARmolecule described herein, is administered at a dose and/or dosingschedule described herein.

In one embodiment, the CAR molecule is introduced into immune effectorcells (e.g., T cells, NK cells), e.g., using in vitro transcription, andthe subject (e.g., human) receives an initial administration of cellscomprising a CAR molecule, and one or more subsequent administrations ofcells comprising a CAR molecule, wherein the one or more subsequentadministrations are administered less than 15 days, e.g., 14, 13, 12,11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days after the previousadministration. In one embodiment, more than one administration of cellscomprising a CAR molecule are administered to the subject (e.g., human)per week, e.g., 2, 3, or 4 administrations of cells comprising a CARmolecule are administered per week. In one embodiment, the subject(e.g., human subject) receives more than one administration of cellscomprising a CAR molecule per week (e.g., 2, 3 or 4 administrations perweek) (also referred to herein as a cycle), followed by a week of noadministration of cells comprising a CAR molecule, and then one or moreadditional administration of cells comprising a CAR molecule (e.g., morethan one administration of the cells comprising a CAR molecule per week)is administered to the subject. In another embodiment, the subject(e.g., human subject) receives more than one cycle of cells comprising aCAR molecule, and the time between each cycle is less than 10, 9, 8, 7,6, 5, 4, or 3 days. In one embodiment, the cells comprising a CARmolecule are administered every other day for 3 administrations perweek. In one embodiment, the cells comprising a CAR molecule areadministered for at least two, three, four, five, six, seven, eight ormore weeks.

In one embodiment, the cells expressing a CAR molecule, e.g., a CARmolecule described herein, are administered as a first line treatmentfor the disease, e.g., the cancer, e.g., the cancer described herein. Inanother embodiment, the cells expressing a CAR molecule, e.g., a CARmolecule described herein, are administered as a second, third, fourthline treatment for the disease, e.g., the cancer, e.g., the cancerdescribed herein.

In one embodiment, a population of cells described herein isadministered.

In another aspect, the invention pertains to the isolated nucleic acidmolecule encoding a CAR of the invention, the isolated polypeptidemolecule of a CAR of the invention, the vector comprising a CAR of theinvention, and the cell comprising a CAR of the invention for use as amedicament.

In another aspect, the invention pertains to a the isolated nucleic acidmolecule encoding a CAR of the invention, the isolated polypeptidemolecule of a CAR of the invention, the vector comprising a CAR of theinvention, and the cell comprising a CAR of the invention for use in thetreatment of a disease expressing a cancer associated antigen asdescribed herein.

In another aspect, the invention pertains to a cell expressing a CARmolecule described herein for use as a medicament in combination with acytokine, e.g., IL-7, IL-15 and/or IL-21 as described herein. In anotheraspect, the invention pertains to a cytokine described herein for use asa medicament in combination with a cell expressing a CAR moleculedescribed herein.

In another aspect, the invention pertains to a cell expressing a CARmolecule described herein for use as a medicament in combination with akinase inhibitor and/or a checkpoint inhibitor as described herein. Inanother aspect, the invention pertains to a kinase inhibitor and/or acheckpoint inhibitor described herein for use as a medicament incombination with a cell expressing a CAR molecule described herein.

In another aspect, the invention pertains to a cell expressing a CARmolecule described herein for use in combination with a cytokine, e.g.,IL-7, IL-15 and/or IL-21 as described herein, in the treatment of adisease expressing a tumor antigen targeted by the CAR. In anotheraspect, the invention pertains to a cytokine described herein for use incombination with a cell expressing a CAR molecule described herein, inthe treatment of a disease expressing a tumor antigen targeted by theCAR.

In another aspect, the invention pertains to a cell expressing a CARmolecule described herein for use in combination with a kinase inhibitorand/or a checkpoint inhibitor as described herein, in the treatment of adisease expressing a tumor antigen targeted by the CAR. In anotheraspect, the invention pertains to a kinase inhibitor and/or a checkpointinhibitor described herein for use in combination with a cell expressinga CAR molecule described herein, in the treatment of a diseaseexpressing a tumor antigen targeted by the CAR.

In another aspect, the present invention provides a method comprisingadministering a CAR molecule, e.g., a CAR molecule described herein, ora cell comprising a nucleic acid encoding a CAR molecule, e.g., a CARmolecule described herein. In one embodiment, the subject has a disorderdescribed herein, e.g., the subject has cancer, e.g., the subject has acancer and has tumor-supporting cells which express a tumor-supportingantigen described herein. In one embodiment, the subject is a human.

In another aspect, the invention pertains to a method of treating asubject having a disease associated with expression of atumor-supporting antigen as described herein comprising administering tothe subject an effective amount of a cell comprising a CAR molecule,e.g., a CAR molecule described herein.

In yet another aspect, the invention features a method of treating asubject having a disease associated with expression of atumor-supporting antigen, comprising administering to the subject aneffective amount of a cell, e.g., an immune effector cell (e.g., apopulation of immune effector cells) comprising a CAR molecule, whereinthe CAR molecule comprises an antigen binding domain, a transmembranedomain, and an intracellular domain, said intracellular domain comprisesa costimulatory domain and/or a primary signaling domain, wherein saidantigen binding domain binds to the tumor-supporting antigen associatedwith the disease, e.g. a tumor-supporting antigen as described herein.

In another aspect, the invention features a composition comprising animmune effector cell (e.g., a population of immune effector cells)comprising a CAR molecule (e.g., a CAR molecule as described herein) foruse in the treatment of a subject having a disease associated withexpression of a tumor-supporting antigen, e.g., a disorder as describedherein.

In any of the aforesaid methods or uses, the disease associated withexpression of the tumor-supporting antigen is selected from the groupconsisting of a proliferative disease, a precancerous condition, acancer, and a non-cancer related indication associated with expressionof the tumor-supporting antigen. In an embodiment, the diseaseassociated with a tumor-supporting antigen described herein is a solidtumor.

In one embodiment of the methods or uses described herein, the CARmolecule is administered in combination with another agent. In oneembodiment, the agent can be a kinase inhibitor, e.g., a CDK4/6inhibitor, a BTK inhibitor, an mTOR inhibitor, a MNK inhibitor, or adual PI3K/mTOR inhibitor, and combinations thereof. In one embodiment,the kinase inhibitor is a CDK4 inhibitor, e.g., a CDK4 inhibitordescribed herein, e.g., a CD4/6 inhibitor, such as, e.g.,6-Acetyl-8-cyclopentyl-5-methyl-2-(5-piperazin-1-yl-pyridin-2-ylamino)-8H-pyrido[2,3-d]pyrimidin-7-one,hydrochloride (also referred to as palbociclib or PD0332991). In oneembodiment, the kinase inhibitor is a BTK inhibitor, e.g., a BTKinhibitor described herein, such as, e.g., ibrutinib. In one embodiment,the kinase inhibitor is an mTOR inhibitor, e.g., an mTOR inhibitordescribed herein, such as, e.g., rapamycin, a rapamycin analog, OSI-027.The mTOR inhibitor can be, e.g., an mTORC1 inhibitor and/or an mTORC2inhibitor, e.g., an mTORC1 inhibitor and/or mTORC2 inhibitor describedherein. In one embodiment, the kinase inhibitor is a MNK inhibitor,e.g., a MNK inhibitor described herein, such as, e.g.,4-amino-5-(4-fluoroanilino)-pyrazolo[3,4-d] pyrimidine. The MNKinhibitor can be, e.g., a MNK1a, MNK1b, MNK2a and/or MNK2b inhibitor.The dual PI3K/mTOR inhibitor can be, e.g., PF-04695102.

In one embodiment of the methods or uses described herein, the kinaseinhibitor is a CDK4 inhibitor selected from aloisine A; flavopiridol orHMR-1275,2-(2-chlorophenyl)-5,7-dihydroxy-8-[(3S,4R)-3-hydroxy-1-methyl-4-piperidinyl]-4-chromenone;crizotinib (PF-02341066;2-(2-Chlorophenyl)-5,7-dihydroxy-8-[(2R,3S)-2-(hydroxymethyl)-1-methyl-3-pyrrolidinyl]-4H-1-benzopyran-4-one,hydrochloride (P276-00);1-methyl-5-[[2-[5-(trifluoromethyl)-1H-imidazol-2-yl]-4-pyridinyl]oxy]-N-[4-(trifluoromethyl)phenyl]-1H-benzimidazol-2-amine(RAF265); indisulam (E7070); roscovitine (CYC202); palbociclib(PD0332991); dinaciclib (SCH727965);N-[5-[[(5-tert-butyloxazol-2-yl)methyl]thio]thiazol-2-yl]piperidine-4-carboxamide(BMS 387032);4-[[9-chloro-7-(2,6-difluorophenyl)-5H-pyrimido[5,4-d][2]benzazepin-2-yl]amino]-benzoicacid (MLN8054);5-[3-(4,6-difluoro-1H-benzimidazol-2-yl)-1H-indazol-5-yl]-N-ethyl-4-methyl-3-pyridinemethanamine(AG-024322); 4-(2,6-dichlorobenzoylamino)-1H-pyrazole-3-carboxylic acidN-(piperidin-4-yl)amide (AT7519);4-[2-methyl-1-(1-methylethyl)-1H-imidazol-5-yl]-N-[4-(methylsulfonyl)phenyl]-2-pyrimidinamine(AZD5438); and XL281 (BMS908662).

In one embodiment of the methods or uses described herein, the kinaseinhibitor is a CDK4 inhibitor, e.g., palbociclib (PD0332991), and thepalbociclib is administered at a dose of about 50 mg, 60 mg, 70 mg, 75mg, 80 mg, 90 mg, 100 mg, 105 mg, 110 mg, 115 mg, 120 mg, 125 mg, 130mg, 135 mg (e.g., 75 mg, 100 mg or 125 mg) daily for a period of time,e.g., daily for 14-21 days of a 28 day cycle, or daily for 7-12 days ofa 21 day cycle. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12or more cycles of palbociclib are administered.

In one embodiment of the methods or uses described herein, the kinaseinhibitor is a BTK inhibitor selected from ibrutinib (PCI-32765);GDC-0834; RN-486; CGI-560; CGI-1764; HM-71224; CC-292; ONO-4059;CNX-774; and LFM-A13. In one embodiment, the BTK inhibitor does notreduce or inhibit the kinase activity of interleukin-2-inducible kinase(ITK), and is selected from GDC-0834; RN-486; CGI-560; CGI-1764;HM-71224; CC-292; ONO-4059; CNX-774; and LFM-A13.

In one embodiment of the methods or uses described herein, the kinaseinhibitor is a BTK inhibitor, e.g., ibrutinib (PCI-32765), and theibrutinib is administered at a dose of about 250 mg, 300 mg, 350 mg, 400mg, 420 mg, 440 mg, 460 mg, 480 mg, 500 mg, 520 mg, 540 mg, 560 mg, 580mg, 600 mg (e.g., 250 mg, 420 mg or 560 mg) daily for a period of time,e.g., daily for 21 day cycle, or daily for 28 day cycle. In oneembodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles ofibrutinib are administered.

In one embodiment of the methods or uses described herein, the kinaseinhibitor is a BTK inhibitor that does not inhibit the kinase activityof ITK, e.g., RN-486, and RN-486 is administered at a dose of about 100mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg (e.g., 150 mg, 200 mgor 250 mg) daily for a period of time, e.g., daily a 28 day cycle. Inone embodiment, 1, 2, 3, 4, 5, 6, 7, or more cycles of RN-486 areadministered.

In one embodiment of the methods or uses described herein, the kinaseinhibitor is an mTOR inhibitor selected from temsirolimus; ridaforolimus(1R,2R,4S)-4-[(2R)-2 [(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28Z,30S,32S,35R)-1,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-2,3,10,14,20-pentaoxo-11,36-dioxa-4-azatricyclo[30.3.1.0^(4,9)]hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyl dimethylphosphinate, also known as AP23573 and MK8669; everolimus(RAD001); rapamycin (AY22989); simapimod;(5-{2,4-bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl}-2-methoxyphenyl)methanol(AZD8055);2-amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one(PF04691502); andN²-[1,4-dioxo-4-[[4-(4-oxo-8-phenyl-4H-1-benzopyran-2-yl)morpholinium-4-yl]methoxy]butyl]-L-arginylglycyl-L-α-aspartylL-serine-,inner salt (SF1126); and XL765.

In one embodiment of the methods or uses described herein, the kinaseinhibitor is an mTOR inhibitor, e.g., rapamycin, and the rapamycin isadministered at a dose of about 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9mg, 10 mg (e.g., 6 mg) daily for a period of time, e.g., daily for 21day cycle, or daily for 28 day cycle. In one embodiment, 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12 or more cycles of rapamycin are administered. Inone embodiment, the kinase inhibitor is an mTOR inhibitor, e.g.,everolimus and the everolimus is administered at a dose of about 2 mg,2.5 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg,13 mg, 14 mg, 15 mg (e.g., 10 mg) daily for a period of time, e.g.,daily for 28 day cycle. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12 or more cycles of everolimus are administered.

In one embodiment of the methods or uses described herein, the kinaseinhibitor is an MNK inhibitor selected from CGP052088;4-amino-3-(p-fluorophenylamino)-pyrazolo[3,4-d] pyrimidine (CGP57380);cercosporamide; ETC-1780445-2; and4-amino-5-(4-fluoroanilino)-pyrazolo[3,4-d] pyrimidine.

In one embodiment of the methods or uses described herein, the kinaseinhibitor is a dual phosphatidylinositol 3-kinase (PI3K) and mTORinhibitor selected from2-Amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one(PF-04691502);N-[4-[[4-(Dimethylamino)-1-piperidinyl]carbonyl]phenyl]-N-[4-(4,6-di-4-morpholinyl-1,3,5-triazin-2-yl)phenyl]urea(PF-05212384, PKI-587);2-Methyl-2-{4-[3-methyl-2-oxo-8-(quinolin-3-yl)-2,3-dihydro-1H-imidazo[4,5-c]quinolin-1-yl]phenyl}propanenitrile(BEZ-235); apitolisib (GDC-0980, RG7422);2,4-Difluoro-N-{2-(methyloxy)-5-[4-(4-pyridazinyl)-6-quinolinyl]-3-pyridinyl}benzenesulfonamide(GSK2126458);8-(6-methoxypyridin-3-yl)-3-methyl-1-(4-(piperazin-1-yl)-3-(trifluoromethyl)phenyl)-1H-imidazo[4,5-c]quinolin-2(3H)-oneMaleic acid (NVP-BGT226);3-[4-(4-Morpholinylpyrido[3′,2′:4,5]furo[3,2-d]pyrimidin-2-yl]phenol(PI-103);5-(9-isopropyl-8-methyl-2-morpholino-9H-purin-6-yl)pyrimidin-2-amine(VS-5584, SB2343); andN-[2-[(3,5-Dimethoxyphenyl)amino]quinoxalin-3-yl]-4-[(4-methyl-3-methoxyphenyl)carbonyl]aminophenylsulfonamide(XL765).

In one embodiment of the methods or uses described herein, a CARexpressing immune effector cell described herein is administered to asubject in combination with a protein tyrosine phosphatase inhibitor,e.g., a protein tyrosine phosphatase inhibitor described herein. In oneembodiment, the protein tyrosine phosphatase inhibitor is an SHP-1inhibitor, e.g., an SHP-1 inhibitor described herein, such as, e.g.,sodium stibogluconate. In one embodiment, the protein tyrosinephosphatase inhibitor is an SHP-2 inhibitor.

In one embodiment of the methods or uses described herein, the CARmolecule is administered in combination with another agent, and theagent is a cytokine. The cytokine can be, e.g., IL-7, IL-15, IL-21, or acombination thereof. In another embodiment, the CAR molecule isadministered in combination with a checkpoint inhibitor, e.g., acheckpoint inhibitor described herein. For example, in one embodiment,the check point inhibitor inhibits an inhibitory molecule selected fromPD-1, PD-L1, CTLA-4, TIM-3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/orCEACAM-5), LAG-3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFR beta.

In one aspect, the CAR of the invention can be used to eradicate anormal cell that express a tumor antigen as described herein, therebyapplicable for use as a cellular conditioning therapy prior to celltransplantation. In one aspect, the normal cell that expresses a tumorantigen as described herein is a normal stem cell and the celltransplantation is a stem cell transplantation.

Therapeutic Application

In another aspect, a method of treating a subject, e.g., reducing orameliorating, a hyperproliferative condition or disorder (e.g., acancer), e.g., solid tumor, a soft tissue tumor, or a metastatic lesion,in a subject is provided. As used herein, the term “cancer” is meant toinclude all types of cancerous growths or oncogenic processes,metastatic tissues or malignantly transformed cells, tissues, or organs,irrespective of histopathologic type or stage of invasiveness. Examplesof solid tumors include malignancies, e.g., sarcomas, adenocarcinomas,and carcinomas, of the various organ systems, such as those affectingliver, lung, breast, lymphoid, gastrointestinal (e.g., colon),genitourinary tract (e.g., renal, urothelial cells), prostate andpharynx. Adenocarcinomas include malignancies such as most coloncancers, rectal cancer, renal-cell carcinoma, liver cancer, non-smallcell carcinoma of the lung, cancer of the small intestine and cancer ofthe esophagus. In one embodiment, the cancer is a melanoma, e.g., anadvanced stage melanoma. Metastatic lesions of the aforementionedcancers can also be treated or prevented using the methods andcompositions of the invention. Examples of other cancers that can betreated include bone cancer, pancreatic cancer, skin cancer, cancer ofthe head or neck, cutaneous or intraocular malignant melanoma, uterinecancer, ovarian cancer, rectal cancer, cancer of the anal region,stomach cancer, testicular cancer, uterine cancer, carcinoma of thefallopian tubes, carcinoma of the endometrium, carcinoma of the cervix,carcinoma of the vagina, carcinoma of the vulva, Hodgkin Disease,non-Hodgkin lymphoma, cancer of the esophagus, cancer of the smallintestine, cancer of the endocrine system, cancer of the thyroid gland,cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma ofsoft tissue, cancer of the urethra, cancer of the penis, chronic oracute leukemias including acute myeloid leukemia, chronic myeloidleukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia,solid tumors of childhood, lymphocytic lymphoma, cancer of the bladder,cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasmof the central nervous system (CNS), primary CNS lymphoma, tumorangiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma,Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T-celllymphoma, environmentally induced cancers including those induced byasbestos, and combinations of said cancers. Treatment of metastaticcancers, e.g., metastatic cancers that express PD-L1 (Iwai et al. (2005)Int. Immunol. 17:133-144) can be effected using the antibody moleculesdescribed herein.

Exemplary cancers whose growth can be inhibited include cancerstypically responsive to immunotherapy. Non-limiting examples of cancersfor treatment include melanoma (e.g., metastatic malignant melanoma),renal cancer (e.g. clear cell carcinoma), prostate cancer (e.g. hormonerefractory prostate adenocarcinoma), breast cancer, colon cancer andlung cancer (e.g. non-small cell lung cancer). Additionally, refractoryor recurrent malignancies can be treated using the molecules describedherein.

In one aspect, the invention pertains to a vector comprising a CARoperably linked to promoter for expression in mammalian immune effectorcells (e.g., T cells, NK cells). In one aspect, the invention provides arecombinant immune effector cell expressing a CAR of the presentinvention for use in treating cancer expressing a cancer associateantigen as described herein. In one aspect, CAR-expressing cells of theinvention is capable of contacting a tumor cell with at least one cancerassociated antigen expressed on its surface such that the CAR-expressingcell targets the cancer cell and growth of the cancer is inhibited.

In one aspect, the invention pertains to a method of inhibiting growthof a cancer, comprising contacting the cancer cell with a CAR-expressingcell of the present invention such that the CART is activated inresponse to the antigen and targets the cancer cell, wherein the growthof the tumor is inhibited.

In one aspect, the invention pertains to a method of treating cancer ina subject. The method comprises administering to the subjectCAR-expressing cell of the present invention such that the cancer istreated in the subject. In one aspect, the cancer associated withexpression of a cancer associate antigen as described herein is ahematological cancer. In one aspect, the hematological cancer is aleukemia or a lymphoma. In one aspect, a cancer associated withexpression of a cancer associate antigen as described herein includescancers and malignancies including, but not limited to, e.g., one ormore acute leukemias including but not limited to, e.g., B-cell acuteLymphoid Leukemia (“BALL”), T-cell acute Lymphoid Leukemia (“TALL”),acute lymphoid leukemia (ALL); one or more chronic leukemias includingbut not limited to, e.g., chronic myelogenous leukemia (CML), ChronicLymphoid Leukemia (CLL). Additional cancers or hematologic conditionsassociated with expression of a cancer associate antigen as describedherein include, but are not limited to, e.g., B cell prolymphocyticleukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt'slymphoma, diffuse large B cell lymphoma, Follicular lymphoma, Hairy cellleukemia, small cell- or a large cell-follicular lymphoma, malignantlymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma,Marginal zone lymphoma, multiple myeloma, myelodysplasia andmyelodysplastic syndrome, non-Hodgkin lymphoma, plasmablastic lymphoma,plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia, and“preleukemia” which are a diverse collection of hematological conditionsunited by ineffective production (or dysplasia) of myeloid blood cells,and the like. Further a disease associated with a cancer associateantigen as described herein expression include, but not limited to,e.g., atypical and/or non-classical cancers, malignancies, precancerousconditions or proliferative diseases associated with expression of acancer associate antigen as described herein.

In some embodiments, a cancer that can be treated with CAR-expressingcell of the present invention is multiple myeloma. Generally, myelomacells are thought to be negative for a cancer associate antigen asdescribed herein expression by flow cytometry. Thus, in someembodiments, a CD19 CAR, e.g., as described herein, may be used totarget myeloma cells. In some embodiments, cars of the present inventiontherapy can be used in combination with one or more additionaltherapies, e.g., lenalidomide treatment.

The invention includes a type of cellular therapy where immune effectorcells (e.g., T cells, NK cells) are genetically modified to express achimeric antigen receptor (CAR) and the CAR-expressing T cell or NK cellis infused to a recipient in need thereof. The infused cell is able tokill tumor cells in the recipient. Unlike antibody therapies,CAR-modified immune effector cells (e.g., T cells, NK cells) are able toreplicate in vivo resulting in long-term persistence that can lead tosustained tumor control. In various aspects, the immune effector cells(e.g., T cells, NK cells) administered to the patient, or their progeny,persist in the patient for at least four months, five months, sixmonths, seven months, eight months, nine months, ten months, elevenmonths, twelve months, thirteen months, fourteen month, fifteen months,sixteen months, seventeen months, eighteen months, nineteen months,twenty months, twenty-one months, twenty-two months, twenty-threemonths, two years, three years, four years, or five years afteradministration of the T cell or NK cell to the patient.

The invention also includes a type of cellular therapy where immuneeffector cells (e.g., T cells, NK cells) are modified, e.g., by in vitrotranscribed RNA, to transiently express a chimeric antigen receptor(CAR) and the CAR T cell or NK cell is infused to a recipient in needthereof. The infused cell is able to kill tumor cells in the recipient.Thus, in various aspects, the immune effector cells (e.g., T cells, NKcells) administered to the patient, is present for less than one month,e.g., three weeks, two weeks, one week, after administration of the Tcell or NK cell to the patient.

Without wishing to be bound by any particular theory, the anti-tumorimmunity response elicited by the CAR-modified immune effector cells(e.g., T cells, NK cells) may be an active or a passive immune response,or alternatively may be due to a direct vs indirect immune response. Inone aspect, the CAR transduced immune effector cells (e.g., T cells, NKcells) exhibit specific proinflammatory cytokine secretion and potentcytolytic activity in response to human cancer cells expressing the acancer associate antigen as described herein, resist soluble a cancerassociate antigen as described herein inhibition, mediate bystanderkilling and mediate regression of an established human tumor. Forexample, antigen-less tumor cells within a heterogeneous field of acancer associate antigen as described herein-expressing tumor may besusceptible to indirect destruction by a cancer associate antigen asdescribed herein-redirected immune effector cells (e.g., T cells, NKcells) that have previously reacted against adjacent antigen-positivecancer cells.

In one aspect, the fully-human CAR-modified immune effector cells (e.g.,T cells, NK cells) of the invention may be a type of vaccine for ex vivoimmunization and/or in vivo therapy in a mammal. In one aspect, themammal is a human.

With respect to ex vivo immunization, at least one of the followingoccurs in vitro prior to administering the cell into a mammal: i)expansion of the cells, ii) introducing a nucleic acid encoding a CAR tothe cells or iii) cryopreservation of the cells.

Ex vivo procedures are well known in the art and are discussed morefully below. Briefly, cells are isolated from a mammal (e.g., a human)and genetically modified (i.e., transduced or transfected in vitro) witha vector expressing a CAR disclosed herein. The CAR-modified cell can beadministered to a mammalian recipient to provide a therapeutic benefit.The mammalian recipient may be a human and the CAR-modified cell can beautologous with respect to the recipient. Alternatively, the cells canbe allogeneic, syngeneic or xenogeneic with respect to the recipient.

The procedure for ex vivo expansion of hematopoietic stem and progenitorcells is described in U.S. Pat. No. 5,199,942, incorporated herein byreference, can be applied to the cells of the present invention. Othersuitable methods are known in the art, therefore the present inventionis not limited to any particular method of ex vivo expansion of thecells. Briefly, ex vivo culture and expansion of immune effector cells(e.g., T cells, NK cells) comprises: (1) collecting CD34+ hematopoieticstem and progenitor cells from a mammal from peripheral blood harvest orbone marrow explants; and (2) expanding such cells ex vivo. In additionto the cellular growth factors described in U.S. Pat. No. 5,199,942,other factors such as flt3-L, IL-1, IL-3 and c-kit ligand, can be usedfor culturing and expansion of the cells.

In addition to using a cell-based vaccine in terms of ex vivoimmunization, the present invention also provides compositions andmethods for in vivo immunization to elicit an immune response directedagainst an antigen in a patient.

Generally, the cells activated and expanded as described herein may beutilized in the treatment and prevention of diseases that arise inindividuals who are immunocompromised. In particular, the CAR-modifiedimmune effector cells (e.g., T cells, NK cells) of the invention areused in the treatment of diseases, disorders and conditions associatedwith expression of a cancer associate antigen as described herein. Incertain aspects, the cells of the invention are used in the treatment ofpatients at risk for developing diseases, disorders and conditionsassociated with expression of a cancer associate antigen as describedherein. Thus, the present invention provides methods for the treatmentor prevention of diseases, disorders and conditions associated withexpression of a cancer associate antigen as described herein comprisingadministering to a subject in need thereof, a therapeutically effectiveamount of the CAR-modified immune effector cells (e.g., T cells, NKcells) of the invention.

In one aspect the CAR-expressing cells of the inventions may be used totreat a proliferative disease such as a cancer or malignancy or is aprecancerous condition such as a myelodysplasia, a myelodysplasticsyndrome or a preleukemia. Further a disease associated with a cancerassociate antigen as described herein expression include, but notlimited to, e.g., atypical and/or non-classical cancers, malignancies,precancerous conditions or proliferative diseases expressing a cancerassociated antigen as described herein. Non-cancer related indicationsassociated with expression of a cancer associate antigen as describedherein include, but are not limited to, e.g., autoimmune disease, (e.g.,lupus), inflammatory disorders (allergy and asthma) and transplantation.

The CAR-modified immune effector cells (e.g., T cells, NK cells) of thepresent invention may be administered either alone, or as apharmaceutical composition in combination with diluents and/or withother components such as IL-2 or other cytokines or cell populations.

Hematologic Cancer

Hematological cancer conditions are the types of cancer such asleukemia, lymphoma, and malignant lymphoproliferative conditions thataffect blood, bone marrow and the lymphatic system.

Leukemia can be classified as acute leukemia and chronic leukemia. Acuteleukemia can be further classified as acute myelogenous leukemia (AML)and acute lymphoid leukemia (ALL). Chronic leukemia includes chronicmyelogenous leukemia (CML) and chronic lymphoid leukemia (CLL). Otherrelated conditions include myelodysplastic syndromes (MDS, formerlyknown as “preleukemia”) which are a diverse collection of hematologicalconditions united by ineffective production (or dysplasia) of myeloidblood cells and risk of transformation to AML.

Lymphoma is a group of blood cell tumors that develop from lymphocytes.Exemplary lymphomas include non-Hodgkin lymphoma and Hodgkin lymphoma.

The present invention also provides methods for inhibiting theproliferation or reducing a cancer associated antigen as describedherein-expressing cell population, the methods comprising contacting apopulation of cells comprising a cancer associated antigen as describedherein-expressing cell with a CAR-expressing T cell or NK cell of theinvention that binds to the a cancer associate antigen as describedherein-expressing cell. In a specific aspect, the present inventionprovides methods for inhibiting the proliferation or reducing thepopulation of cancer cells expressing a cancer associated antigen asdescribed herein, the methods comprising contacting a cancer associateantigen as described herein-expressing cancer cell population with aCAR-expressing T cell or NK cell of the invention that binds to a cancerassociated antigen as described herein-expressing cell. In one aspect,the present invention provides methods for inhibiting the proliferationor reducing the population of cancer cells expressing a cancerassociated antigen as described herein, the methods comprisingcontacting a cancer associated antigen as described herein-expressingcancer cell population with a CAR-expressing T cell or NK cell of theinvention that binds to a cancer associated antigen as describedherein-expressing cell. In certain aspects, a CAR-expressing T cell orNK cell of the invention reduces the quantity, number, amount orpercentage of cells and/or cancer cells by at least 25%, at least 30%,at least 40%, at least 50%, at least 65%, at least 75%, at least 85%, atleast 95%, or at least 99% in a subject with or animal model for myeloidleukemia or another cancer associated with a cancer associated antigenas described herein-expressing cells relative to a negative control. Inone aspect, the subject is a human.

The present invention also provides methods for preventing, treatingand/or managing a disease associated with a cancer associated antigen asdescribed herein-expressing cells (e.g., a hematologic cancer oratypical cancer expressing a cancer associated antigen as describedherein), the methods comprising administering to a subject in need a CART cell or NK cell of the invention that binds to a cancer associatedantigen as described herein-expressing cell. In one aspect, the subjectis a human. Non-limiting examples of disorders associated with a cancerassociated antigen as described herein-expressing cells includeautoimmune disorders (such as lupus), inflammatory disorders (such asallergies and asthma) and cancers (such as hematological cancers oratypical cancers expressing a cancer associated antigen as describedherein).

The present invention also provides methods for preventing, treatingand/or managing a disease associated with a cancer associated antigen asdescribed herein-expressing cells, the methods comprising administeringto a subject in need a CAR T cell or NK cell of the invention that bindsto a cancer associated antigen as described herein-expressing cell. Inone aspect, the subject is a human.

The present invention provides methods for preventing relapse of cancerassociated with a cancer associated antigen as describedherein-expressing cells, the methods comprising administering to asubject in need thereof a CAR T cell or NK cell of the invention thatbinds to a cancer associated antigen as described herein-expressingcell. In one aspect, the methods comprise administering to the subjectin need thereof an effective amount of a CAR-expressing T cell or NKcell described herein that binds to a cancer associated antigen asdescribed herein-expressing cell in combination with an effective amountof another therapy.

Pharmaceutical Compositions and Treatments

Pharmaceutical compositions of the present invention may comprise aCAR-expressing cell, e.g., a plurality of CAR-expressing cells, asdescribed herein, in combination with one or more pharmaceutically orphysiologically acceptable carriers, diluents or excipients. Suchcompositions may comprise buffers such as neutral buffered saline,phosphate buffered saline and the like; carbohydrates such as glucose,mannose, sucrose or dextrans, mannitol; proteins; polypeptides or aminoacids such as glycine; antioxidants; chelating agents such as EDTA orglutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.Compositions of the present invention are in one aspect formulated forintravenous administration.

Pharmaceutical compositions of the present invention may be administeredin a manner appropriate to the disease to be treated (or prevented). Thequantity and frequency of administration will be determined by suchfactors as the condition of the patient, and the type and severity ofthe patient's disease, although appropriate dosages may be determined byclinical trials.

In one embodiment, the pharmaceutical composition is substantially freeof, e.g., there are no detectable levels of a contaminant, e.g.,selected from the group consisting of endotoxin, mycoplasma, replicationcompetent lentivirus (RCL), p24, VSV-G nucleic acid, HIV gag, residualanti-CD3/anti-CD28 coated beads, mouse antibodies, pooled human serum,bovine serum albumin, bovine serum, culture media components, vectorpackaging cell or plasmid components, a bacterium and a fungus. In oneembodiment, the bacterium is at least one selected from the groupconsisting of Alcaligenes faecalis, Candida albicans, Escherichia coli,Haemophilus influenza, Neisseria meningitides, Pseudomonas aeruginosa,Staphylococcus aureus, Streptococcus pneumonia, and Streptococcuspyogenes group A.

When “an immunologically effective amount,” “an anti-tumor effectiveamount,” “a tumor-inhibiting effective amount,” or “therapeutic amount”is indicated, the precise amount of the compositions of the presentinvention to be administered can be determined by a physician withconsideration of individual differences in age, weight, tumor size,extent of infection or metastasis, and condition of the patient(subject). It can generally be stated that a pharmaceutical compositioncomprising the immune effector cells (e.g., T cells, NK cells) describedherein may be administered at a dosage of 10⁴ to 10⁹ cells/kg bodyweight, in some instances 10⁵ to 10⁶ cells/kg body weight, including allinteger values within those ranges. T cell compositions may also beadministered multiple times at these dosages. The cells can beadministered by using infusion techniques that are commonly known inimmunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med.319:1676, 1988).

In certain aspects, it may be desired to administer activated immuneeffector cells (e.g., T cells, NK cells) to a subject and thensubsequently redraw blood (or have an apheresis performed), activateimmune effector cells (e.g., T cells, NK cells) therefrom according tothe present invention, and reinfuse the patient with these activated andexpanded immune effector cells (e.g., T cells, NK cells). This processcan be carried out multiple times every few weeks. In certain aspects,immune effector cells (e.g., T cells, NK cells) can be activated fromblood draws of from 10 cc to 400 cc. In certain aspects, immune effectorcells (e.g., T cells, NK cells) are activated from blood draws of 20 cc,30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, or 100 cc.

The administration of the subject compositions may be carried out in anyconvenient manner, including by aerosol inhalation, injection,ingestion, transfusion, implantation or transplantation. Thecompositions described herein may be administered to a patient transarterially, subcutaneously, intradermally, intratumorally, intranodally,intramedullary, intramuscularly, by intravenous (i.v.) injection, orintraperitoneally. In one aspect, the T cell compositions of the presentinvention are administered to a patient by intradermal or subcutaneousinjection. In one aspect, the T cell compositions of the presentinvention are administered by i.v. injection. The compositions of immuneeffector cells (e.g., T cells, NK cells) may be injected directly into atumor, lymph node, or site of infection.

In a particular exemplary aspect, subjects may undergo leukapheresis,wherein leukocytes are collected, enriched, or depleted ex vivo toselect and/or isolate the cells of interest, e.g., T cells. These T cellisolates may be expanded by methods known in the art and treated suchthat one or more CAR constructs of the invention may be introduced,thereby creating a CAR T cell of the invention. Subjects in need thereofmay subsequently undergo standard treatment with high dose chemotherapyfollowed by peripheral blood stem cell transplantation. In certainaspects, following or concurrent with the transplant, subjects receivean infusion of the expanded CAR T cells of the present invention. In anadditional aspect, expanded cells are administered before or followingsurgery.

The dosage of the above treatments to be administered to a patient willvary with the precise nature of the condition being treated and therecipient of the treatment. The scaling of dosages for humanadministration can be performed according to art-accepted practices. Thedose for CAMPATH, for example, will generally be in the range 1 to about100 mg for an adult patient, usually administered daily for a periodbetween 1 and 30 days. The preferred daily dose is 1 to 10 mg per dayalthough in some instances larger doses of up to 40 mg per day may beused (described in U.S. Pat. No. 6,120,766).

In one embodiment, the CAR is introduced into immune effector cells(e.g., T cells, NK cells), e.g., using in vitro transcription, and thesubject (e.g., human) receives an initial administration of CAR immuneeffector cells (e.g., T cells, NK cells) of the invention, and one ormore subsequent administrations of the CAR immune effector cells (e.g.,T cells, NK cells) of the invention, wherein the one or more subsequentadministrations are administered less than 15 days, e.g., 14, 13, 12,11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days after the previousadministration. In one embodiment, more than one administration of theCAR immune effector cells (e.g., T cells, NK cells) of the invention areadministered to the subject (e.g., human) per week, e.g., 2, 3, or 4administrations of the CAR immune effector cells (e.g., T cells, NKcells) of the invention are administered per week. In one embodiment,the subject (e.g., human subject) receives more than one administrationof the CAR immune effector cells (e.g., T cells, NK cells) per week(e.g., 2, 3 or 4 administrations per week) (also referred to herein as acycle), followed by a week of no CAR immune effector cells (e.g., Tcells, NK cells) administrations, and then one or more additionaladministration of the CAR immune effector cells (e.g., T cells, NKcells) (e.g., more than one administration of the CAR immune effectorcells (e.g., T cells, NK cells) per week) is administered to thesubject. In another embodiment, the subject (e.g., human subject)receives more than one cycle of CAR immune effector cells (e.g., Tcells, NK cells), and the time between each cycle is less than 10, 9, 8,7, 6, 5, 4, or 3 days. In one embodiment, the CAR immune effector cells(e.g., T cells, NK cells) are administered every other day for 3administrations per week. In one embodiment, the CAR immune effectorcells (e.g., T cells, NK cells) of the invention are administered for atleast two, three, four, five, six, seven, eight or more weeks.

In one aspect, CAR-expressing cells of the present inventions aregenerated using lentiviral viral vectors, such as lentivirus. Cells,e.g., CARTs, generated that way will have stable CAR expression.

In one aspect, CAR-expressing cells, e.g., CARTs, are generated using aviral vector such as a gammaretroviral vector, e.g., a gammaretroviralvector described herein. CARTs generated using these vectors can havestable CAR expression.

In one aspect, CARTs transiently express CAR vectors for 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15 days after transduction. Transient expressionof CARs can be effected by RNA CAR vector delivery. In one aspect, theCAR RNA is transduced into the T cell by electroporation.

A potential issue that can arise in patients being treated usingtransiently expressing CAR immune effector cells (e.g., T cells, NKcells) (particularly with murine scFv bearing CARTs) is anaphylaxisafter multiple treatments.

Without being bound by this theory, it is believed that such ananaphylactic response might be caused by a patient developing humoralanti-CAR response, i.e., anti-CAR antibodies having an anti-IgE isotype.It is thought that a patient's antibody producing cells undergo a classswitch from IgG isotype (that does not cause anaphylaxis) to IgE isotypewhen there is a ten to fourteen day break in exposure to antigen.

If a patient is at high risk of generating an anti-CAR antibody responseduring the course of transient CAR therapy (such as those generated byRNA transductions), CART infusion breaks should not last more than tento fourteen days.

Methods of Making CAR-Expressing Cells

In another aspect, the invention pertains to a method of making a cell(e.g., an immune effector cell or population thereof) comprisingintroducing into (e.g., transducing) a cell, e.g., a T cell or a NK celldescribed herein, with a vector of comprising a nucleic acid encoding aCAR, e.g., a CAR described herein; or a nucleic acid encoding a CARmolecule e.g., a CAR described herein.

The cell in the methods is an immune effector cell (e.g., aT cell or aNK cell, or a combination thereof). In some embodiments, the cell in themethods is diaglycerol kinase (DGK) and/or Ikaros deficient.

In some embodiment, the introducing the nucleic acid molecule encoding aCAR comprises transducing a vector comprising the nucleic acid moleculeencoding a CAR, or transfecting the nucleic acid molecule encoding aCAR, wherein the nucleic acid molecule is an in vitro transcribed RNA.

In some embodiments, the method further comprises:

a. providing a population of immune effector cells (e.g., T cells or NKcells); andb. removing T regulatory cells from the population, thereby providing apopulation of T regulatory-depleted cells;wherein steps a) and b) are performed prior to introducing the nucleicacid encoding the CAR to the population.

In embodiments of the methods, the T regulatory cells comprise CD25+ Tcells, and are removed from the cell population using an anti-CD25antibody, or fragment thereof. The anti-CD25 antibody, or fragmentthereof, can be conjugated to a substrate, e.g., a bead.

In other embodiments, the population of T regulatory-depleted cellsprovided from step (b) contains less than 30%, 25%, 20%, 15%, 10%, 5%,4%, 3%, 2%, 1% of CD25+ cells.

In yet other embodiments, the method further comprises removing cellsfrom the population which express a tumor antigen that does not compriseCD25 to provide a population of T regulatory-depleted and tumor antigendepleted cells prior to introducing the nucleic acid encoding a CAR tothe population. The tumor antigen can be selected from CD19, CD30, CD38,CD123, CD20, CD14 or CD11b, or a combination thereof.

In other embodiments, the method further comprises removing cells fromthe population which express a checkpoint inhibitor, to provide apopulation of T regulatory-depleted and inhibitory molecule depletedcells prior to introducing the nucleic acid encoding a CAR to thepopulation. The checkpoint inhibitor can be chosen from PD-1, LAG-3,TIM3, B7-H1, CD160, P1H, 2B4, CEACAM (e.g., CEACAM-1, CEACAM-3, and/orCEACAM-5), TIGIT, CTLA-4, BTLA, and LAIR1.

Further embodiments disclosed herein encompass providing a population ofimmune effector cells. The population of immune effector cells providedcan be selected based upon the expression of one or more of CD3, CD28,CD4, CD8, CD45RA, and/or CD45RO. In certain embodiments, the populationof immune effector cells provided are CD3+ and/or CD28+.

In certain embodiments of the method, the method further comprisesexpanding the population of cells after the nucleic acid moleculeencoding a CAR has been introduced.

In embodiments, the population of cells is expanded for a period of 8days or less.

In certain embodiments, the population of cells is expanded in culturefor 5 days, and the resulting cells are more potent than the same cellsexpanded in culture for 9 days under the same culture conditions.

In other embodiments, the population of cells is expanded in culture for5 days show at least a one, two, three or four fold increase in celldoublings upon antigen stimulation as compared to the same cellsexpanded in culture for 9 days under the same culture conditions.

In yet other embodiments, the population of cells is expanded in culturefor 5 days, and the resulting cells exhibit higher proinflammatory IFN-γand/or GM-CSF levels, as compared to the same cells expanded in culturefor 9 days under the same culture conditions.

In other embodiments, the population of cells is expanded by culturingthe cells in the presence of an agent that stimulates a CD3/TCR complexassociated signal and/or a ligand that stimulates a costimulatorymolecule on the surface of the cells. The agent can be a bead conjugatedwith anti-CD3 antibody, or a fragment thereof, and/or anti-CD28antibody, or a fragment thereof.

In other embodiments, the population of cells is expanded in anappropriate media that includes one or more interleukin that result inat least a 200-fold, 250-fold, 300-fold, or 350-fold increase in cellsover a 14 day expansion period, as measured by flow cytometry.

In other embodiments, the population of cells is expanded in thepresence IL-15 and/or IL-7. In certain embodiments, the method furtherincludes cryopreserving the population of the cells after theappropriate expansion period.

In yet other embodiments, the method of making disclosed herein furthercomprises contacting the population of immune effector cells with anucleic acid encoding a telomerase subunit, e.g., hTERT. The the nucleicacid encoding the telomerase subunit can be DNA.

The present invention also provides a method of generating a populationof RNA-engineered cells, e.g., cells described herein, e.g., immuneeffector cells (e.g., T cells, NK cells), transiently expressingexogenous RNA. The method comprises introducing an in vitro transcribedRNA or synthetic RNA into a cell, where the RNA comprises a nucleic acidencoding a CAR molecule described herein.

In another aspect, the invention pertains to a method of providing ananti-tumor immunity in a subject comprising administering to the subjectan effective amount of a cell comprising a CAR molecule, e.g., a cellexpressing a CAR molecule described herein. In one embodiment, the cellis an autologous T cell or NK cell. In one embodiment, the cell is anallogeneic T cell or NK cell. In one embodiment, the subject is a human.

In one aspect, the invention includes a population of autologous cellsthat are transfected or transduced with a vector comprising a nucleicacid molecule encoding a CAR molecule, e.g., as described herein. In oneembodiment, the vector is a retroviral vector. In one embodiment, thevector is a self-inactivating lentiviral vector as described elsewhereherein. In one embodiment, the vector is delivered (e.g., bytransfecting or electroporating) to a cell, e.g., a T cell or a NK cell,wherein the vector comprises a nucleic acid molecule encoding a CAR ofthe present invention as described herein, which is transcribed as anmRNA molecule, and the CARs of the present invention is translated fromthe RNA molecule and expressed on the surface of the cell.

In another aspect, the present invention provides a population ofCAR-expressing cells, e.g., CAR-expressing immune effector cells (e.g.,T cells or NK cells). In some embodiments, the population ofCAR-expressing cells comprises a mixture of cells expressing differentCARs. For example, in one embodiment, the population of CAR-expressingimmune effector cells (e.g., T cells or NK cells) can include a firstcell expressing a CAR having an antigen binding domain that binds to afirst tumor antigen as described herein, and a second cell expressing aCAR having a different antigen binding domain that binds to a secondtumor antigen as described herein. As another example, the population ofCAR-expressing cells can include a first cell expressing a CAR thatincludes an antigen binding domain that binds to a tumor antigen asdescribed herein, and a second cell expressing a CAR that includes anantigen binding domain to a target other than a tumor antigen asdescribed herein. In one embodiment, the population of CAR-expressingcells includes, e.g., a first cell expressing a CAR that includes aprimary intracellular signaling domain, and a second cell expressing aCAR that includes a secondary signaling domain, e.g., a costimulatorysignaling domain.

In another aspect, the present invention provides a population of cellswherein at least one cell in the population expresses a CAR having anantigen binding domain that binds to a tumor antigen as describedherein, and a second cell expressing another agent, e.g., an agent whichenhances the activity of a CAR-expressing cell. For example, in oneembodiment, the agent can be an agent which inhibits an inhibitorymolecule. Examples of inhibitory molecules include PD-1, PD-L1, CTLA-4,TIM-3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG-3, VISTA,BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFR beta. In one embodiment, theagent which inhibits an inhibitory molecule, e.g., is a moleculedescribed herein, e.g., an agent that comprises a first polypeptide,e.g., an inhibitory molecule, associated with a second polypeptide thatprovides a positive signal to the cell, e.g., an intracellular signalingdomain described herein. In one embodiment, the agent comprises a firstpolypeptide, e.g., of an inhibitory molecule such as PD-1, LAG-3,CTLA-4, CD160, BTLA, LAIR1, TIM-3, CEACAM (e.g., CEACAM-1, CEACAM-3and/or CEACAM-5), 2B4 and TIGIT, or a fragment of any of these, and asecond polypeptide which is an intracellular signaling domain describedherein (e.g., comprising a costimulatory domain (e.g., 41 BB, CD27 orCD28, e.g., as described herein) and/or a primary signaling domain(e.g., a CD3 zeta signaling domain described herein). In one embodiment,the agent comprises a first polypeptide of PD-1 or a fragment thereof,and a second polypeptide of an intracellular signaling domain describedherein (e.g., a CD28, CD27, OX40 or 4-IBB signaling domain describedherein and/or a CD3 zeta signaling domain described herein).

In one embodiment, the nucleic acid molecule encoding a CAR of thepresent invention molecule, e.g., as described herein, is expressed asan mRNA molecule. In one embodiment, the genetically modified CAR of thepresent invention-expressing cells, e.g., immune effector cells (e.g., Tcells, NK cells), can be generated by transfecting or electroporating anRNA molecule encoding the desired CARs (e.g., without a vector sequence)into the cell. In one embodiment, a CAR of the present inventionmolecule is translated from the RNA molecule once it is incorporated andexpressed on the surface of the recombinant cell.

A method for generating mRNA for use in transfection involves in vitrotranscription (IVT) of a template with specially designed primers,followed by polyA addition, to produce a construct containing 3′ and 5′untranslated sequence (“UTR”) (e.g., a 3′ and/or 5′ UTR describedherein), a 5′ cap (e.g., a 5′ cap described herein) and/or InternalRibosome Entry Site (IRES) (e.g., an IRES described herein), the nucleicacid to be expressed, and a polyA tail, typically 50-2000 bases inlength. RNA so produced can efficiently transfect different kinds ofcells. In one embodiment, the template includes sequences for the CAR.In an embodiment, an RNA CAR vector is transduced into a cell, e.g., a Tcell or a NK cell, by electroporation.

Sequences of some examples of various components of CARs of the instantinvention is listed in Table 1, where aa stands for amino acids, and nastands for nucleic acids that encode the corresponding peptide.

TABLE 1Sequences of various components of CAR (aa-amino acids, na-nucleic acids thatencodes the corresponding protein) SEQ Corresp. ID To NO description Sequence huCD19 1 EF-1CGTGAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAA 100 promoterGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGA 2 Leader (aa)  MALPVTALLLPLALLLHAARP 13 3Leader (na)  ATGGCCCTGCCTGTGACAGCCCTGCTGCTGCCTCTGGCTCTGCTGCTGCATGCCGCTA54 GACCC 4 CD 8 hinge TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD 14(aa) 5 CD8 hingeACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCC 55 (na)TGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT 6 Ig4 hingeESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFN 102 (aa)WYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKM 7 Ig4 hingeGAGAGCAAGTACGGCCCTCCCTGCCCCCCTTGCCCTGCCCCCGAGTTCCTGGGCGGAC 103 (na)CCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAGGTGACCTGTGTGGTGGTGGACGTGTCCCAGGAGGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCCGGGAGGAGCAGTTCAATAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGTAAGGTGTCCAACAAGGGCCTGCCCAGCAGCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTCGGGAGCCCCAGGTGTACACCCTGCCCCCTAGCCAAGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCCGGCTGACCGTGGACAAGAGCCGGTGGCAGGAGGGCAACGTCTTTAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCTGGGCAAGATG 8 IgD hingeRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERET 47 (aa)KTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDH 9 IgD hingeAGGTGGCCCGAAAGTCCCAAGGCCCAGGCATCTAGTGTTCCTACTGCACAGCCCCAGG 48 (na)CAGAAGGCAGCCTAGCCAAAGCTACTACTGCACCTGCCACTACGCGCAATACTGGCCGTGGCGGGGAGGAGAAGAAAAAGGAGAAAGAGAAAGAAGAACAGGAAGAGAGGGAGACCAAGACCCCTGAATGTCCATCCCATACCCAGCCGCTGGGCGTCTATCTCTTGACTCCCGCAGTACAGGACTTGTGGCTTAGAGATAAGGCCACCTTTACATGTTTCGTCGTGGGCTCTGACCTGAAGGATGCCCATTTGACTTGGGAGGTTGCCGGAAAGGTACCCACAGGGGGGGTTGAGGAAGGGTTGCTGGAGCGCCATTCCAATGGCTCTCAGAGCCAGCACTCAAGACTCACCCTTCCGAGATCCCTGTGGAACGCCGGGACCTCTGTCACATGTACTCTAAATCATCCTAGCCTGCCCCCACAGCGTCTGATGGCCCTTAGAGAGCCAGCCGCCCAGGCACCAGTTAAGCTTAGCCTGAATCTGCTCGCCAGTAGTGATCCCCCAGAGGCCGCCAGCTGGCTCTTATGCGAAGTGTCCGGCTTTAGCCCGCCCAACATCTTGCTCATGTGGCTGGAGGACCAGCGAGAAGTGAACACCAGCGGCTTCGCTCCAGCCCGGCCCCCACCCCAGCCGGGTTCTACCACATTCTGGGCCTGGAGTGTCTTAAGGGTCCCAGCACCACCTAGCCCCCAGCCAGCCACATACACCTGTGTTGTGTCCCATGAAGATAGCAGGACCCTGCTAAATGCTTCTAGGAGTCTGGAGGTTTCCTACGTGACTGACCATTGGGGSGGGGS 10 GS GGGGSGGGGS 49hinge/linker (aa) 11 GS GGTGGCGGAGGTTCTGGAGGTGGAGGTTCC 50 hinge/linker(na) 12 CD8TM (aa) IYIWAPLAGTCGVLLLSLVITLYC 15 13 CD8 TMATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTA 56 (na)TCACCCTTTACTGC 14 4-1BB KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL 16intracellular domain (aa) 15 4-1BBAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTAC 60intracellular AAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGdomain (na) ATGTGAACTG 16 CD27 (aa)QRRKYRSNKGESPVEPAEPCRYSCPREEEGSTIPIQEDYRKPEPACSP 51 17 CD27 (na)AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCC 52CCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTA TCGCTCC 18CD3-zeta RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGL 17(aa) YNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 19 CD3-zetaAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGC 101 (na)TCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC 20 CD3-zetaRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGL 43 (aa)YNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 21 CD3-zetaAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGC 44 (na)TCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC 22 linker GGGGS 18 23linker GGTGGCGGAGGTTCTGGAGGTGGAGGTTCC 50 24 PD-1pgwfldspdrpwnpptfspallvvtegdnatftcsfsntsesfvlnwyrmspsnqtdkextracellular laafpedrsqpgqdcrfrvtqlpngrdfhmsvvrarrndsgtylcgaislapkaqike domain (aa)slraelrvterraevptahpspsprpagqfqtlv 25 PD-1cccggatggtttctggactctccggatcgcccgtggaatcccccaaccttctcaccggextracellular cactcttggttgtgactgagggcgataatgcgaccttcacgtgctcgttctccaacac domain (na)ctccgaatcattcgtgctgaactggtaccgcatgagcccgtcaaaccagaccgacaagctcgccgcgtttccggaagatcggtcgcaaccgggacaggattgtcggttccgcgtgactcaactgccgaatggcagagacttccacatgagcgtggtccgcgctaggcgaaacgactccgggacctacctgtgcggagccatctcgctggcgcctaaggcccaaatcaaagagagcttgagggccgaactgagagtgaccgagcgcagagctgaggtgccaactgcacatccatccccatcgcctcggcctgcggggcagtttcagaccctggtc 26 PD-1 CARmalpvtalllplalllhaarppgwfldspdrpwnpptfspallvvtegdnatftcsfs (aa) withntsesfvlnwyrmspsnqtdklaafpedrsqpgqdcrfrvtqlpngrdfhmsvvrarr signalndsgtylcgaislapkaqikeslraelryterraevptahpspsprpagqfqtivtttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr 27 PD-1 CARatggccctccctgtcactgccctgcttctccccctcgcactcctgctccacgccgcta (na)gaccacccggatggtttctggactctccggatcgcccgtggaatcccccaaccttctcaccggcactcttggttgtgactgagggcgataatgcgaccttcacgtgctcgttctccaacacctccgaatcattcgtgctgaactggtaccgcatgagcccgtcaaaccagaccgacaagctcgccgcgtttccggaagatcggtcgcaaccgggacaggattgtcggttccgcgtgactcaactgccgaatggcagagacttccacatgagcgtggtccgcgctaggcgaaacgactccgggacctacctgtgcggagccatctcgctggcgcctaaggcccaaatcaaagagagcttgagggccgaactgagagtgaccgagcgcagagctgaggtgccaactgcacatccatccccatcgcctcggcctgcggggcagtttcagaccctggtcacgaccactccggcgccgcgcccaccgactccggccccaactatcgcgagccagcccctgtcgctgaggccggaagcatgccgccctgccgccggaggtgctgtgcatacccggggattggacttcgcatgcgacatctacatttgggctcctctcgccggaacttgtggcgtgctccttctgtccctggtcatcaccctgtactgcaagcggggtcggaaaaagcttctgtacattttcaagcagcccttcatgaggcccgtgcaaaccacccaggaggaggacggttgctcctgccggttccccgaagaggaagaaggaggttgcgagctgcgcgtgaagttctcccggagcgccgacgcccccgcctataagcagggccagaaccagctgtacaacgaactgaacctgggacggcgggaagagtacgatgtgctggacaagcggcgcggccgggaccccgaaatgggcgggaagcctagaagaaagaaccctcaggaaggcctgtataacgagctgcagaaggacaagatggccgaggcctactccgaaattgggatgaagggagagcggcggaggggaaaggggcacgacggcctgtaccaaggactgtccaccgccaccaaggacacatacgatgccctgcacatgcaggcccttccccctcgc 28 linker (Gly-Gly-Gly-Ser)n,  105 where n = 1-1029 linker (Gly4 Ser)4 106 30 linker (Gly4 Ser)3 107 31 linker (Gly3Ser)108 32 PD1CAR powfldspdrpwnpptfspallvvtegdnatftcsfsntsesfvlnwyrmspsnqtdk(aa) laafpedrsqpoqdcrfrvtqlpnordfhmsvvrarrndsotylcgaislapkaqikeslraelrvterraevptahpspsprpagqfqtlvtttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvIllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr 33 CD19 CARMALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQ (aa) murineQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 34 CD19 CAR atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgcca (na) murineggccggacatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagggcaagtcaggacattagtaaatatttaaattggtatcagcagaaaccagatggaactgttaaactcctgatctaccatacatcaagattacactcaggagtcccatcaaggttcagtggcagtgggtctggaacagattattctctcaccattagcaacctggagcaagaagatattgccacttacttttgccaacagggtaatacgcttccgtacacgttcggaggggggaccaagctggagatcacaggtggcggtggctcgggcggtggtgggtcgggtggcggcggatctgaggtgaaactgcaggagtcaggacctggcctggtggcgccctcacagagcctgtccgtcacatgcactgtctcaggggtctcattacccgactatggtgtaagctggattcgccagcctccacgaaagggtctggagtggctgggagtaatatggggtagtgaaaccacatactataattcagctctcaaatccagactgaccatcatcaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccatttactactgtgccaaacattattactacggtggtagctatgctatggactactggggccaaggaacctcagtcaccgtctcctcaaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccgcgtacaagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgc cccctcgct 35CD19 CAR MALPVTALLLPLALLLHAARPEIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQ(aa) human QKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQGTKLEIKGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYQSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 36 CD19 CARatggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgctc (na) humanggcccgaaattgtgatgacccagtcacccgccactcttagcctttcacccggtgagcgcgcaaccctgtcttgcagagcctcccaagacatctcaaaataccttaattggtatcaacagaagcccggacaggctcctcgccttctgatctaccacaccagccggctccattctggaatccctgccaggttcagcggtagcggatctgggaccgactacaccctcactatcagctcactgcagccagaggacttcgctgtctatttctgtcagcaagggaacaccctgccctacacctttggacagggcaccaagctcgagattaaaggtggaggtggcagcggaggaggtgggtccggcggtggaggaagccaggtccaactccaagaaagcggaccgggtcttgtgaagccatcagaaactctttcactgacttgtactgtgagcggagtgtctctccccgattacggggtgtcttggatcagacagccaccggggaagggtctggaatggattggagtgatttggggctctgagactacttactaccaatcatccctcaagtcacgcgtcaccatctcaaaggacaactctaagaatcaggtgtcactgaaactgtcatctgtgaccgcagccgacaccgccgtgtactattgcgctaagcattactattatggcgggagctacgcaatggattactggggacagggtactctggtcaccgtgtccagcaccactaccccagcaccgaggccacccaccccggctcctaccatcgcctcccagcctctgtccctgcgtccggaggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgc cgcctcgg

TABLE 2 Antigen Binding domains that bind B cell antigens B cell SEQ IDantigen Name Amino Acid Sequence NO: CD19 huscFEIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYH 37 v1TSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQGTKLEIKGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYSSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSS CD19 huscFEIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYH 38 v2TSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQGTKLEIKGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYQSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSS CD19 huscFQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGV 39 v3IWGSETTYYSSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQGTKLEIK CD19 huscFQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGV 40 v4IWGSETTYYQSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQGTKLEIK CD19 huscFEIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYH 41 v5TSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQGTKLEIKGGGGSGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYSSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSS CD19 huscFEIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYH 42 v6TSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQGTKLEIKGGGGSGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYQSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSS CD19 huscFQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGV 43 v7IWGSETTYYSSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQGTKLEIK CD19 huscFQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGV 44 v8IWGSETTYYQSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQGTKLEIK CD19 huscFEIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYH 45 v9TSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQGTKLEIKGGGGSGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYNSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSS CD19 HuQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGV 46 scFv10IWGSETTYYNSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQGTKLEIK CD19 HuEIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYH 47 scFv11TSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQGTKLEIKGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYNSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSS CD19 HuQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGV 48 scFv12IWGSETTYYNSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQGTKLEIK CD19 muCTDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYH 49 L019TSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS

The following examples are intended to illustrate, rather than limit,the invention.

EXAMPLES Example 1. Generation of the pCINS Vector

The parental lentiviral transfer vector pRRL.SIN.cPPT.EF1a.EGFP.WPRE wassynthesized de novo by DNA2.0 using sequences derived from the backboneof pRRL.SIN vector made by Dull et al. (J. Virol. 72: 8463-8471, 1998;incorporated herein by reference). A feature map of pCINS is set forthin FIG. 1, while a restriction map of the vector is set forth in FIG. 2.

In generating the pCINS vector, the following modifications were made tothe parental vector:

-   -   1. 5′ cis elements were replaced with the corresponding natural        sequence from HIV-1 isolate NL4-3. The replaced 5′ cis elements        included: packaging signal (psi), partial gag sequence adjacent        to psi, Rev-response element (RRE) and the partial env sequence        surrounding it, and a central polypurine tract (cPPT) sequence        from pol.    -   2. The SV40 and f1 origins of replication were removed as        redundant to decrease plasmid size.    -   3. Several restriction sites were introduced between the cis        elements to facilitate DNA engineering.    -   4. The INS1 inhibitory sequence in gag, which restricts nuclear        export of unspliced viral RNA (J. Virol. 71(7):4892-4903,        1997; J. Virol. 66(12):7176-7182, 1992; each of which is        incorporated herein by reference), was mutated, thereby reducing        restriction of nuclear export of viral RNA(s) encoded by the        vector.    -   5. Part of the gag sequence, after nucleotide 168, which        contains inhibitory sequences INS2, INS3, and INS4 (J. Virol.        68(6):3784-3793, 1994; incorporated herein by reference), was        removed.    -   6. The RSV promoter was replaced with a CMV promoter, since both        RSV and CMV promoter can be used for SIN LV expression (Science        272(5259):263-267, 1996; J. Virol. 72(11):8463-8471, 1998; each        of which is incorporated herein by reference).

The pCINS-EGFP sequence is set forth below and is detailed in Table 3.EGFP sequences can optionally be replaced with sequences of anothertransgene (e.g., a gene encoding a CAR), if desired, using standardmethods in the art.

pCINS-EGFP sequence  (SEQ ID NO: 50)gcagactagtaagcttagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatgctgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctggtttagtgaaccggggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtggcgcccgaacagggacttgaaagcgaaagtaaagccagaggagatctctcgacgcaggactcggcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaaaattttgactagcggaggctagaaggagagagatgggtgcgagagcgtcggtattaagcgggggagaattagataaatgggaaaaaattcggtaataaggccagggggaaagaagaagtacaagctaaagcacatcgtatgggcaagcagggagctagaacgattcgcagttaatcctggccttttagagacatcagaaggcggccgctgatcttcagacctggaggaggcgatatgagggacaattggagaagtgaattatataaatataaagtagtaaaaattgaaccattaggagtagcacccaccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaatttaaataggagctttgttccttgggttcttgggagcagcaggaagcactatgggcgcagcgtcaatgacgctgacggtacaggccagacaattattgtctgatatagtgcagcagcagaacaatttgctgagggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaaacagctccaggcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctcctgcaggggatttggggttgctctggaaaactcatttgcaccactgctgtgccttggaatgctagttggagtaataaatctctggaacagatttggaataacatgacctggatggagtgggacagagaaattaacaattacacaagcttaatacactccttaattgaagaatcgcaaaaccagcaagaaaagaatgaacaagaattattggaattagataaatgggcaagtttgtggaattggtttaacataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggtttaagaatagtttttgctgtactttctatagtgaatagagttaggcagggatattcaccattatcgtttcagacccacctcccaatcccgaggggacccgacaggcccgaaggaatagaagaagaaggtggagagagagacagagacagatccattcgattagtgaacggatctcgacggtatcgattagactgtagcccaggaatatggcagctagattgtacacatttagaaggaaaagttatcttggtagcagttcatgtagccagtggatatatagaagcagaagtaattccagcagagacagggcaagaaacagcatacttcctcttaaaattagcaggaagatggccagtaaaaacagtacatacagacaatggcagcaatttcaccagtactacagttaaggccgcctgttggtgggcggggatcaagcaggaatttggcattccctacaatccccaaagtcaaggagtaatagaatctatgaataaagaattaaagaaaattataggacaggtaagagatcaggctgaacatcttaagacagcagtacaaatggcagtattcatccacaattttaaaagaaaaggggggattggggggtacagtgcaggggaaagaatagtagacataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaattttcgggtttattacagggacagcagagatccagtttggctgcattgatcacgtgaggctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtcggcaattgaaccggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcctttttcccgagggtgggggagaaccgtatataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgccgccagaacacaggtaagtgccgtgtgtggttcccgcgggcctggcctctttacgggttatggcccttgcgtgccttgaattacttccacctggctgcagtacgtgattcttgatcccgagcttcgggttggaagtgggtgggagagttcgaggccttgcgcttaaggagccccttcgcctcgtgcttgagttgaggcctggcctgggcgctggggccgccgcgtgcgaatctggtggcaccttcgcgcctgtctcgctgctttcgataagtctctagccatttaaaatttttgatgacctgctgcgacgctttttttctggcaagatagtcttgtaaatgcgggccaagatctgcacactggtatttcggtttttggggccgcgggcggcgacggggcccgtgcgtcccagcgcacatgttcggcgaggcggggcctgcgagcgcggccaccgagaatcggacgggggtagtctcaagctggccggcctgctctggtgcctggcctcgcgccgccgtgtatcgccccgccctgggcggcaaggctggcccggtcggcaccagttgcgtgagcggaaagatggccgcttcccggccctgctgcagggagctcaaaatggaggacgcggcgctcgggagagcgggcgggtgagtcacccacacaaaggaaaagggcctttccgtcctcagccgtcgcttcatgtgactccactgagtaccgggcgccgtccaggcacctcgattagttctcgagcttttggagtacgtcgtctttaggttggggggaggggttttatgcgatggagtttccccacactgagtgggtggagactgaagttaggccagcttggcacttgatgtaattctccttggaatttgccctttttgagtttggatcttggttcattctcaagcctcagacagtggttcaaagtttttttcttccatttcaggtgtcgtgatctagaggatccgccaccatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaagtcgacaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaagctgacgtcctttccatggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgcctggaattcgagctcggtacctttaagaccaatgacttacaaggcagctgtagatcttagccactttttaaaagaaaaggggggactggaagggctaattcactcccaacgaagacaagatctgctttttgcttgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtagtagttcatgtcatcttattattcagtatttataacttgcaaagaaatgaatatcagagagtgagaggaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctggctctagctatcccgcccctaggcaccggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagccatattcaacgggaaacgtcgaggccgcgattaaattccaacatggatgctgatttatatgggtataaatgggctcgcgataatgtcgggcaatcaggtgcgacaatctatcgcttgtatgggaagcccgatgcgccagagttgtttctgaaacatggcaaaggtagcgttgccaatgatgttacagatgagatggtcagactaaactggctgacggaatttatgccacttccgaccatcaagcattttatccgtactcctgatgatgcatggttactcaccactgcgatccccggaaaaacagcgttccaggtattagaagaatatcctgattcaggtgaaaatattgttgatgcgctggcagtgttcctgcgccggttgcactcgattcctgtttgtaattgtccttttaacagcgatcgcgtatttcgcctcgctcaggcgcaatcacgaatgaataacggtttggttgatgcgagtgattttgatgacgagcgtaatggctggcctgttgaacaagtctggaaagaaatgcataaacttttgccattctcaccggattcagtcgtcactcatggtgatttctcacttgataaccttatttttgacgaggggaaattaataggttgtattgatgttggacgagtcggaatcgcagaccgataccaggatcttgccatcctatggaactgcctcggtgagttttctccttcattacagaaacggctttttcaaaaatatggtattgataatcctgatatgaataaattgcagtttcatttgatgctcgatgagtttttctaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagcgcgcaattaaccctcactaaagggaacaaaagctggagct

Example 2. Generation of the pNOX Vector

The pCINS vector generated in Example 1 was further modified to producethe pNOX vector. In particular, the post-transcriptional regulatoryelement (PRE) of woodchuck hepatitis virus (WPRE) present in pCINS wasreplaced with a PRE from hepatitis B virus (HPRE), which includes thenatural sequence of hepatitis B virus isolate bba6, complete genome(GenBank: KP341007.1). WPRE was present in the parental vector to ensureefficient expression of the transgene. However, WPRE contains an Xprotein-coding sequence. The presence of the X protein ORF in WPRE maypose safety issues for integrating lentiviral vectors. For example, Xprotein has been implicated in the generation of liver cancers (GeneTher. 12(1):3-4, 2005; incorporated herein by reference). In the pNOXvector, a point mutation was introduced in the start codon (ATG->AGG) ofthe X protein. As a result, the recombinant HPRE contains no X proteinORF. A feature map of pNOX is set forth in FIG. 13, while a restrictionmap of the vector is set forth in FIG. 14.

The pNOX-EGFP sequence is set forth below and is detailed in Table 4.EGFP sequences can optionally be replaced with sequences of anothertransgene (e.g., a gene encoding a CAR), if desired, using standardmethods in the art.

pNOX-EGFP sequence  (SEQ ID NO: 51)gcagactagtaagcttagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatgctgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctggtttagtgaaccggggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtggcgcccgaacagggacttgaaagcgaaagtaaagccagaggagatctctcgacgcaggactcggcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaaaattttgactagcggaggctagaaggagagagatgggtgcgagagcgtcggtattaagcgggggagaattagataaatgggaaaaaattcggtaataaggccagggggaaagaagaagtacaagctaaagcacatcgtatgggcaagcagggagctagaacgattcgcagttaatcctggccttttagagacatcagaaggcggccgctgatcttcagacctggaggaggcgatatgagggacaattggagaagtgaattatataaatataaagtagtaaaaattgaaccattaggagtagcacccaccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaatttaaataggagctttgttccttgggttcttgggagcagcaggaagcactatgggcgcagcgtcaatgacgctgacggtacaggccagacaattattgtctgatatagtgcagcagcagaacaatttgctgagggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaaacagctccaggcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctcctgcaggggatttggggttgctctggaaaactcatttgcaccactgctgtgccttggaatgctagttggagtaataaatctctggaacagatttggaataacatgacctggatggagtgggacagagaaattaacaattacacaagcttaatacactccttaattgaagaatcgcaaaaccagcaagaaaagaatgaacaagaattattggaattagataaatgggcaagtttgtggaattggtttaacataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggtttaagaatagtttttgctgtactttctatagtgaatagagttaggcagggatattcaccattatcgtttcagacccacctcccaatcccgaggggacccgacaggcccgaaggaatagaagaagaaggtggagagagagacagagacagatccattcgattagtgaacggatctcgacggtatcgattagactgtagcccaggaatatggcagctagattgtacacatttagaaggaaaagttatcttggtagcagttcatgtagccagtggatatatagaagcagaagtaattccagcagagacagggcaagaaacagcatacttcctcttaaaattagcaggaagatggccagtaaaaacagtacatacagacaatggcagcaatttcaccagtactacagttaaggccgcctgttggtgggcggggatcaagcaggaatttggcattccctacaatccccaaagtcaaggagtaatagaatctatgaataaagaattaaagaaaattataggacaggtaagagatcaggctgaacatcttaagacagcagtacaaatggcagtattcatccacaattttaaaagaaaaggggggattggggggtacagtgcaggggaaagaatagtagacataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaattttcgggtttattacagggacagcagagatccagtttggctgcattgatcacgtgaggctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtcggcaattgaaccggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcctttttcccgagggtgggggagaaccgtatataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgccgccagaacacaggtaagtgccgtgtgtggttcccgcgggcctggcctctttacgggttatggcccttgcgtgccttgaattacttccacctggctgcagtacgtgattcttgatcccgagcttcgggttggaagtgggtgggagagttcgaggccttgcgcttaaggagccccttcgcctcgtgcttgagttgaggcctggcctgggcgctggggccgccgcgtgcgaatctggtggcaccttcgcgcctgtctcgctgctttcgataagtctctagccatttaaaatttttgatgacctgctgcgacgctttttttctggcaagatagtcttgtaaatgcgggccaagatctgcacactggtatttcggtttttggggccgcgggcggcgacggggcccgtgcgtcccagcgcacatgttcggcgaggcggggcctgcgagcgcggccaccgagaatcggacgggggtagtctcaagctggccggcctgctctggtgcctggcctcgcgccgccgtgtatcgccccgccctgggcggcaaggctggcccggtcggcaccagttgcgtgagcggaaagatggccgcttcccggccctgctgcagggagctcaaaatggaggacgcggcgctcgggagagcgggcgggtgagtcacccacacaaaggaaaagggcctttccgtcctcagccgtcgcttcatgtgactccactgagtaccgggcgccgtccaggcacctcgattagttctcgagcttttggagtacgtcgtctttaggttggggggaggggttttatgcgatggagtttccccacactgagtgggtggagactgaagttaggccagcttggcacttgatgtaattctccttggaatttgccctttttgagtttggatcttggttcattctcaagcctcagacagtggttcaaagtttttttcttccatttcaggtgtcgtgatctagaggatccgccaccatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaagtcgactaaacaggcctattgattggaaagtatgtcaacgaattgtgggtcttttggggtttgctgccccttttacgcaatgtggatatcctgctttaatgcctttatatgcatgtatacaagcaaaacaggcttttactttctcgccaacttacaaggcctttctaagtaaacagtatctgaccctttaccccgttgctcggcaacggcctggtctgtgccaagtgtttgctgacgcaacccccactggttggggcttggccataggccatcagcgcatgcgtggaacctttgtgtctcctctgccgatccatactgcggaactcctagccgcttgttttgctcgcagcaggtctggagcgaaactcatcgggactgacaattctgtcgtgctctcccgcaagtatacatcgtttccagggctgctaggctgtgctgccaactggatcctgcgcgggacgtcctttgtttacgtcccgtcggcgctgaatcccgcggacgacccctcccggggccgcttggggctctaccgcccgcttctccgtctgccgtaccgaccgaccacggggcgcacctctctttacgcggactccccgtctgtgccttctcatctgccggaccgtgtgcacttcgcttcacctctgcacgtcgcatggagaccaccgtgaacgcccaccggaacctgcccaaggtcttgcataagaggactcttggactttcagcaatgtcaacgaattcgagctcggtacctttaagaccaatgacttacaaggcagctgtagatcttagccactttttaaaagaaaaggggggactggaagggctaattcactcccaacgaagacaagatctgctttttgcttgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtagtagttcatgtcatcttattattcagtatttataacttgcaaagaaatgaatatcagagagtgagaggaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctggctctagctatcccgcccctaggcaccggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagccatattcaacgggaaacgtcgaggccgcgattaaattccaacatggatgctgatttatatgggtataaatgggctcgcgataatgtcgggcaatcaggtgcgacaatctatcgcttgtatgggaagcccgatgcgccagagttgtttctgaaacatggcaaaggtagcgttgccaatgatgttacagatgagatggtcagactaaactggctgacggaatttatgccacttccgaccatcaagcattttatccgtactcctgatgatgcatggttactcaccactgcgatccccggaaaaacagcgttccaggtattagaagaatatcctgattcaggtgaaaatattgttgatgcgctggcagtgttcctgcgccggttgcactcgattcctgtttgtaattgtccttttaacagcgatcgcgtatttcgcctcgctcaggcgcaatcacgaatgaataacggtttggttgatgcgagtgattttgatgacgagcgtaatggctggcctgttgaacaagtctggaaagaaatgcataaacttttgccattctcaccggattcagtcgtcactcatggtgatttctcacttgataaccttatttttgacgaggggaaattaataggttgtattgatgttggacgagtcggaatcgcagaccgataccaggatcttgccatcctatggaactgcctcggtgagttttctccttcattacagaaacggctttttcaaaaatatggtattgataatcctgatatgaataaattgcagtttcatttgatgctcgatgagtttttctaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagcgcgcaattaaccctcactaaagggaacaaaagctggagct

Example 3. Viral Titers Generated Using the pCINS and pNOX Vectors

To determine the efficacy of the pCINS and pNOX lentiviral transfervectors, a series of experiments was performed, in which one of thetransfer vectors was introduced into cells using transient transfectionof packaging cells. Briefly, Expi293™ cells (ThermoFischer) were grownin 5 ml of Freestyle (FS) medium at concentration 5 min/ml at 200 rpm,37° C. and 8% CO2, 80% humidity. Transfection was performed using PEIProTransfer Reagent (PolyPlus) using 3 μg pNVS-MDLgp-RRE, 3 μg pNVS RSVRev-Kan, 0.75 μg pNVS-MDG-VSVG-Kan, and 6 μg transfer vector (pCINS orpNOX). Viral supernatants were collected 48 hours after transfection andsubjected to titer analysis (i.e., measurement of infectious titer).

In 293T cells, the pCINS plasmid transfer vector generated a viral titerapproximately five times higher than that generated by the parentalvector (FIG. 5A). Titer was evaluated based on percentage ofGFP-positive cells. This unexpectedly strong viral production may be dueto higher quantities of lentiviral genomic RNA generated by CMVpromoter-mediated transcription in packaging cells (measured by qRT-PCR)and more efficient export of unspliced LV RNA from nucleus due to theabsence of nuclear retention.

The pNOX transfer vector was able to generate similar or higher vectortiters compared to pCINS (FIG. 5B-5D). Infectious titers were measuredby GFP expression in transduced 293T cells, Jurkat T cells, and primaryhuman T cells. In particular, pNOX generated similar levels of GFPexpression to pCINS in 293T cells (FIG. 5B) and in primary human T cells(FIG. 5D), but actually yielded substantially greater quantities ofGFP-expressing Jurkat cells relative to pCINS (FIG. 5C).

The level of transgene expression after genomic integration of elementsfrom a lentiviral transfer vector was also examined. T cells wereinfected with viruses produced using the pNOX or pCINS transfer vectors,such that viral elements were integrated into the T cell genomes. TheHPRE-containing lentiviral vector, pNOX, resulted in similar levels oftransgene expressed compared to the WPRE-containing lentiviral vector,pCINS (FIG. 5 E). These results were surprising based on previousreports that WPRE (in pCINS) is more potent than HPRE (see, e.g., J.Virol. 72: 5085-5092, 1998; Gene Therapy 14, 1298-1304, 2007; each ofwhich is incorporated herein by reference).

The invention includes the pCINS vector, as well as related vectors thatinclude portions of the pCINS vector and/or sequences that shareidentity (e.g., at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identitywith pCINS) with one or more sequences of pCINS. Sequences that may beincluded in such related vectors include all pCINS sequences, or subsetsincluding, e.g., various combinations of the viral promoter (i.e., thepromoter driving expression of the viral proteins), partial gag (e.g.,lacking INS2, 3, and 4, and/or including an INS1 mutation as describedherein), partial env, RRE, cPPT, subgenomic promoter (e.g., EF1alphapromoter, optionally including constitutive splice donor and spliceacceptor sites as described herein), and PRE (optionally with an Xprotein inactivating mutation, as described herein) (these sequenceseach optionally have the sequence identities noted above). Such vectorsmay include a transgene (e.g., a gene encoding a CAR or EGFP, asdescribed herein).

TABLE 3  pCINS Features SEQ Feature Nucleic Acid Sequence ID NO CMVGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACA 52 promoterTAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGCTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCG RGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGA 53ACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTC U5AAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACC 54CTTTTAGTCAGTGTGGAAAATCTCTAGCAG PBS TGGCGCCCGAACAGGGAC 55 PackagingTTGAAAGCGAAAGTAAAGCCAGAGGAGATCTCTCGACGCAGGACTCGGCTTGCTG 56 signalAAGCGCGCACGGCAAGAGGCGAGGGGCGGCG ACTGGTGAGT ACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCGGTATTA SDACTGGTGAGT (indicated in underlining in packaging 57signal sequence above) Partial gagATGGGTGCGAGAGCGTCGGTATTAAGCGGGGGAGAATTAGATAAATG 58 sequenceGGAAAAAATTCGGTAATAAGGCCAGGGGGAAAGAAGAAGTACAAGCT (from NL4-3) AAAGCACATCGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTA with mutated ATCCTGGCCTTTTAGAGACATCAGAAG INS signal NotI GCGGCCGC 59 restriction sitePartial env TGATCTTCAGACCTGGAGGAGGCGATATGAGGGACAATTGGAGAAGT 60 sequenceGAATTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGT GGGA SwaI ATTTAAAT 61restriction site RREAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCG 62TCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGATATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAACAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAG GATCAACAGCTCCTSbfI ATTTAAAT 63 restriction site Partial envGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCT 64 sequenceTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATAAC containingATGACCTGGATGGAGTGGGACAGAGAAATTAACAATTACACAAGCTT spliceAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAAGAAAAGAATGA acceptorACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTT SA7TAACATAACAAATTGGCTGTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTG AATAGAGTTAGGCAGGGATATTCACCATTATCGTTTCAGAC CCACCTCCCAATCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATC TCGACGGT SA7AGTTAGGCAGGGATATTCACCATTATCGTTTCAGAC 65 ClaI ATCGAT 66 restriction sitecPPT TAGACTGTAGCCCAGGAATATGGCAGCTAGATTGTACACATTTAGAAGGAAAAGT 67TATCTTGGTAGCAGTTCATGTAGCCAGTGGATATATAGAAGCAGAAGTAATTCCAGCAGAGACAGGGCAAGAAACAGCATACTTCCTCTTAAAATTAGCAGGAAGATGGCCAGTAAAAACAGTACATACAGACAATGGCAGCAATTTCACCAGTACTACAGTTAAGGCCGCCTGTTGGTGGGCGGGGATCAAGCAGGAATTTGGCATTCCCTACAATCCCCAAAGTCAAGGAGTAATAGAATCTATGAATAAAGAATTAAAGAAAATTATAGGACAGGTAAGAGATCAGGCTGAACATCTTAAGACAGCAGTACAAATGGCAGTATTCATCCACAATTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTCGGGTTTATTACAGGGACAGCAGAGATCCAGTTTGGCT SA1GTTTATTACAGGGACAGCAGAGATCCAGTTTGG  68(indicated in underlining in cppt sequence above) Blunted PstI  CTGCAT 69 restriction site BclI TGATCA 70 restriction site EF1aCGTGAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGA 71 promoterGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACTGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGA Constitutive  CAGAACACAGGTAAGTGCCG 72splice donor  (indicated by underlining in P-EF1a sequence above) (CD)Constitutive TCCATTTCAGGTGTCGTGA 73 splice(indicated by underlining in P-EF1a sequence above) acceptor (CA) XbalTCTAGA 74 restriction site BamHI GGATCC 75 restriction site EGFPATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGC 76TGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACA AGTAA SalIGTCGAC 77 restriction site WPREATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGT 78TGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTG EcoRI GAATTC 80 restrictionsite SacI GAGCTC 81 restriction site KpnI GGTACC 82 restriction sitePartial Nef CTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTT 83sequence, TAAAAGAAAAGGGGGG containing PPT dU3ACTGGAAGGGCTAATTCACTCCCAACGAAGACAAGATCTGCTTTTTGC 84 TTGTACT RGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGA 85ACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTC U5AAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACC 66CTTTTAGTCAGTGTGGAAAATCTCTAGCAG SV40 polyAAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATT 87TCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTC nptlIATGAGCCATATTCAACGGGAAACGTCGAGGCCGCGATTAAATTCCAACATGGATG 88CTGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCTATCGCTTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCACTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCCCCGGAAAAACAGCGTTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCACTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGCCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAACTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAA pUC oriAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCA 89AACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAA AACGCCAGCAACGCG

The invention includes the pNOX vector, as well as related vectors thatinclude portions of the pNOX vector and/or sequences that share identity(e.g., at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity withpNOX) with one or more sequences of pNOX. Sequences that may be includedin such related vectors include all pNOX sequences, or subsetsincluding, e.g., various combinations of the viral promoter (i.e., thepromoter driving expression of the viral proteins), partial gag (e.g.,lacking INS2, 3, and 4, and/or including an INS1 mutation as describedherein), partial env, RRE, cPPT, subgenomic promoter (e.g., EF1alphapromoter, optionally including constitutive splice donor and spliceacceptor sites as described herein), and PRE (optionally with an Xprotein inactivating mutation, as described herein) (these sequenceseach optionally have the sequence identities noted above). Such vectorsmay include a transgene (e.g., a gene encoding a CAR or EGFP, asdescribed herein).

TABLE 4  pNOX Features SEQ Feature Nucleic Acid Sequence ID NO CMVGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACA 52 promoterTAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGCTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCG RGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGA 53ACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTC U5AAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACC 54CTTTTAGTCAGTGTGGAAAATCTCTAGCAG PBS TGGCGCCCGAACAGGGAC 55 PackagingTTGAAAGCGAAAGTAAAGCCAGAGGAGATCTCTCGACGCAGGACTCGGCTTGCTG 56 signalAAGCGCGCACGGCAAGAGGCGAGGGGCGGCG ACTGGTGAGT ACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCGGTATTA SDACTGGTGAGT (indicated in underlining in packaging  57signal sequence above) Partial gagATGGGTGCGAGAGCGTCGGTATTAAGCGGGGGAGAATTAGATAAATGGGAAAAAA 58 sequenceTTCGGTAATAAGGCCAGGGGGAAAGAAGAAGTACAAGCTAAAGCACATCGTATGG (from NL4-3)GCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTTTTAGAGACATCAG with mutated AAGINS signal NotI GCGGCCGC 59 restriction site Partial envTGATCTTCAGACCTGGAGGAGGCGATATGAGGGACAATTGGAGAAGTGAATTATA 60 sequenceTAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGA SwaI ATTTAAAT 61 restriction siteRRE AGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCG 62TCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGATATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAACAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAG GATCAACAGCTCCTSbfI CCTGCAGG 63 restriction site Partial env GGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCT 64 sequenceTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATAAC containingATGACCTGGATGGAGTGGGACAGAGAAATTAACAATTACACAAGCTT spliceAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAAGAAAAGAATGA acceptorACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTT 5A7TAACATAACAAATTGGCTGTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTG AATAGAGTTAGGCAGGGATATTCACCATTATCGTTTCAGAC CCACCTCCCAATCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATC TCGACGGT SA7AGTTAGGCAGGGATATTCACCATTATCGTTTCAGAC 65 ClaI ATCGAT 66 restriction sitecPPT TAGACTGTAGCCCAGGAATATGGCAGCTAGATTGTACACATTTAGAAGGAAAAGT 67TATCTTGGTAGCAGTTCATGTAGCCAGTGGATATATAGAAGCAGAAGTAATTCCAGCAGAGACAGGGCAAGAAACAGCATACTTCCTCTTAAAATTAGCAGGAAGATGGCCAGTAAAAACAGTACATACAGACAATGGCAGCAATTTCACCAGTACTACAGTTAAGGCCGCCTGTTGGTGGGCGGGGATCAAGCAGGAATTTGGCATTCCCTACAATCCCCAAAGTCAAGGAGTAATAGAATCTATGAATAAAGAATTAAAGAAAATTATAGGACAGGTAAGAGATCAGGCTGAACATCTTAAGACAGCAGTACAAATGGCAGTATTCATCCACAATTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTCGGGTTTATTACAGGGACAGCAGAGATCCAGTTTGGCT SA1GTTTATTACAGGGACAGCAGAGATCCAGTTTGG (indicated in 68underlining in cppt sequence above) Blunted Pstl CTGCAT 69 restrictionsite BclI TGATCA 70 restriction site EF1aCGTGAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGA 71 promoterGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACTGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGA Constitutive  CAGAACACAGGTAAGTGCCG 72splice donor  (indicated by underlining in P-EF1a sequence above) (CD)Constitutive TCCATTTCAGGTGTCGTGA 73 splice(indicated by underlining in P-EF1a sequence above) acceptor (CA) XbaITCTAGA 74 restriction site BamHI GGATCC 75 restriction site EGFPATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGC 76TGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACA AGTAA SalIGTCGAC 77 restriction site HPRE NoXTAAACAGGCCTATTGATTGGAAAGTATGTCAACGAATTGTGGGTCTTTTGGGGTT 79TGCTGCCCCTTTTACGCAATGTGGATATCCTGCTTTAATGCCTTTATATGCATGTATACAAGCAAAACAGGCTTTTACTTTCTCGCCAACTTACAAGGCCTTTCTAAGTAAACAGTATCTGACCCTTTACCCCGTTGCTCGGCAACGGCCTGGTCTGTGCCAAGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCTTGGCCATAGGCCATCAGCGCATGCGTGGAACCTTTGTGTCTCCTCTGCCGATCCATACTGCGGAACTCCTAGCCGCTTGTTTTGCTCGCAGCAGGTCTGGAGCGAAACTCATCGGGACTGACAATTCTGTCGTGCTCTCCCGCAAGTATACATCGTTTCCAGGGCTGCTAGGCTGTGCTGCCAACTGGATCCTGCGCGGGACGTCCTTTGTTTACGTCCCGTCGGCGCTGAATCCCGCGGACGACCCCTCCCGGGGCCGCTTGGGGCTCTACCGCCCGCTTCTCCGTCTGCCGTACCGACCGACCACGGGGCGCACCTCTCTTTACGCGGACTCCCCGTCTGTGCCTTCTCATCTGCCGGACCGTGTGCACTTCGCTTCACCTCTGCACGTCGCATGGAGACCACCGTGAACGCCCACCGGAACCTGCCCAAGGTCTTGCATAAGAGGACTCTTGGACTTTCA GCAATGTCAAC(underlined codon is mutated ATG/AGG codon of X protein ORF) EcoRIGAATTC 80 restriction site SacI GAGCTC 81 restriction site KpnI GGTACC82 restriction site Partial NefCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTT 83 sequence,TAAAAGAAAAGGGGGG containing PPT dU3ACTGGAAGGGCTAATTCACTCCCAACGAAGACAAGATCTGCTTTTTGC 84 TTGTACT RGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGA 85ACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTC U5AAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACC 86CTTTTAGTCAGTGTGGAAAATCTCTAGCAG SV40 polyAAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATT 87TCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTC nptlIATGAGCCATATTCAACGGGAAACGTCGAGGCCGCGATTAAATTCCAACATGGATG 88CTGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCTATCGCTTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCACTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCCCCGGAAAAACAGCGTTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCACTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGCCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAACTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAA pUC onAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCA 89AACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAA AACGCCAGCAACGCG

Example 4. Generation of the pNLV Transfer Vector

The pNOX vector generated in Example 2 was further modified to producethe pNLV vector. Feature and restriction maps of pNLV are shown in FIGS.13 and 14, respectively. The cPPT element of pNOX was replaced with thecPPT sequence shown in SEQ ID NO:92. cPPT represents pol sequenceposition 2698-2850 (see SEQ ID NOs:92 and 93). A Kozak sequence isincluded immediately upstream of the gene encoding the transgene (e.g.,EGFP, as shown in Table 5). Lastly, a wild-type EF1a promoter (P-EF1a)of SEQ ID NO: 95 was utilized. The pNLV vector has increased viral titerand an improved biosafety profile.

The sequence of the pNLV vector, and the sequences of the elements inthe pNLV vector, are shown below. In some instances, the vector backboneand elements included in the pNLV vector, and portions thereof, may beused in any of the vectors described herein. Vector backbone sequencesand functional elements may be inserted into another vector and/orreplace portions of another vector according to cloning methods wellknown in the art.

The pNLV-EGFP sequence is set forth below and is detailed in Table 5.EGFP sequences can optionally be replaced with sequences of anothertransgene (e.g., a gene encoding a CAR), if desired, using standardmethods in the art.

pNLV-EGFP sequence  (SEQ ID NO: 90)GTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGCTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCGGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGTAAAGCCAGAGGAGATCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCGGTATTAAGCGGGGGAGAATTAGATAAATGGGAAAAAATTCGGTAATAAGGCCAGGGGGAAAGAAGAAGTACAAGCTAAAGCACATCGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTTTTAGAGACATCAGAAGGCGGCCGCTGATCTTCAGACCTGGAGGAGGCGATATGAGGGACAATTGGAGAAGTGAATTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATTTAAATAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGATATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAACAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTCCTGCAGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATAACATGACCTGGATGGAGTGGGACAGAGAAATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGCAGGGATATTCACCATTATCGTTTCAGACCCACCTCCCAATCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCTCGACGGTATCGATACTAGTACAAATGGCAGTATTCATCCACAATTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTCGGGTTTATTACAGGGACAGCAGAGATCCACTTTGGCTGCATTGATCACGTGAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGATCTAGAGGATCCGCCACCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAGTCGACTAAACAGGCCTATTGATTGGAAAGTATGTCAACGAATTGTGGGTCTTTTGGGGTTTGCTGCCCCTTTTACGCAATGTGGATATCCTGCTTTAATGCCTTTATATGCATGTATACAAGCAAAACAGGCTTTTACTTTCTCGCCAACTTACAAGGCCTTTCTAAGTAAACAGTATCTGACCCTTTACCCCGTTGCTCGGCAACGGCCTGGTCTGTGCCAAGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCTTGGCCATAGGCCATCAGCGCATGCGTGGAACCTTTGTGTCTCCTCTGCCGATCCATACTGCGGAACTCCTAGCCGCTTGTTTTGCTCGCAGCAGGTCTGGAGCGAAACTCATCGGGACTGACAATTCTGTCGTGCTCTCCCGCAAGTATACATCGTTTCCAGGGCTGCTAGGCTGTGCTGCCAACTGGATCCTGCGCGGGACGTCCTTTGTTTACGTCCCGTCGGCGCTGAATCCCGCGGACGACCCCTCCCGGGGCCGCTTGGGGCTCTACCGCCCGCTTCTCCGTCTGCCGTACCGACCGACCACGGGGCGCACCTCTCTTTACGCGGACTCCCCGTCTGTGCCTTCTCATCTGCCGGACCGTGTGCACTTCGCTTCACCTCTGCACGTCGCATGGAGACCACCGTGAACGCCCACCGGAACCTGCCCAAGGTCTTGCATAAGAGGACTCTTGGACTTTCAGCAATGTCAACGAATTCGAGCTCGGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAACGAAGACAAGATCTGCTTTTTGCTTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTAGTAGTTCATGTCATCTTATTATTCAGTATTTATAACTTGCAAAGAAATGAATATCAGAGAGTGAGAGGAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGCTCTAGCTATCCCGCCCCTAGGCACCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGCCATATTCAACGGGAAACGTCGAGGCCGCGATTAAATTCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCTATCGCTTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCACTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCCCCGGAAAAACAGCGTTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCACTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGCCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAACTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCGCGCAATTAACCCTCACTAAAGGGAACAAAAGCTGGAGCTGCAGACTAGTAAGCTTA

The functional elements present within the pNLV vector are shown inTable 4 below. Portions of the full pNLV sequence may include vectorbackbone sequences. Such vector backbone sequences may be used as and/orreplace vector backbone sequences, or portions thereof, in any of thevectors described herein (e.g., pCINS, pNOX, and pNLV).

The invention includes the pNLV vector, as well as related vectors thatinclude portions of the pNLV vector and/or sequences that share identity(e.g., at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity withpNLV) with one or more sequences of pNLV. Sequences that may be includedin such related vectors include all pNLV sequences, or subsetsincluding, e.g., various combinations of the viral promoter (i.e., thepromoter driving expression of the viral proteins), partial gag (e.g.,lacking INS2, 3, and 4, and/or including an INS1 mutation as describedherein), partial env, RRE, cPPT, subgenomic promoter (e.g., EF1alphapromoter, optionally including constitutive splice donor and spliceacceptor sites as described herein), and PRE (optionally with an Xprotein inactivating mutation, as described herein) (these sequenceseach optionally have the sequence identities noted above). Such vectorsmay include a transgene (e.g., a gene encoding a CAR or EGFP, asdescribed herein).

TABLE 5  pNLV features SEQ Feature Nucleic Acid Sequence Source ID NO:CMV GTAATCAATTACGGGGTCATTAGTTCATAGCCCATA GenBank: 52 promoter/TATGGAGTTCCGCGTTACATAACTTACGGTAAATG KT186368.1 enhancer,GCCCGCCTGGCTGACCGCCCAACGACCCCCGCCC P-CMVATTGACGTCAATAATGACGTATGTTCCCATAGTAAC GCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACAT CAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATG CCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATG CTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCC ACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAA CTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTGGTTT AGTGAACCG RGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGG NL4-3 HIV-1 53AGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAG isolate CCTCAATAAAGCTTGCCTTGAGTGCTTCGenBank: AF324493.1 U5 AAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGT NL4-3 HIV-154 AACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTG isolate GAAAATCTCTAGCAG GenBank:AF324493.1 PBS TGGCGCCCGAACAGGGAC NL4-3 HIV-1 55 isolate GenBank:AF324493.1 Packaging TTGAAAGCGAAAGTAAAGCCAGAGGAGATCTCTCG NL4-3 HIV-1 56signal, psi ACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCA isolate AGAGGCGAGGGGCGGCGACTGGTGAGT ACGCCA GenBank: AAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGAAF324493.1 TGGGTGCGAGAGCGTCGGTATTA Major splice ACTGGTGAGT (indicated by underlining inside 57 donor, SDpackaging signal sequence above) Partial gagATGGGTGCGAGAGCGTCGGTATTAAGCGGGGGAG Modified 58 sequenceAATTAGATAAATGGGAAAAAATTCGGTAATAAGGC sequence (from NL4-3) CAGGGGGAAAGAAGAAGTACAAGCTAAAGCACAT from NL4-3 with mutated CGTATGGGCAAGCAGGGAGCTAGAACGATTCGCA HIV-1 isolate INS signalGTTAATCCTGGCCTTTTAGAGACATCAGAAG GenBank: AF324493.1 NotI GCGGCCGC 59restriction site Partial env TGATCTTCAGACCTGGAGGAGGCGATATGAGGGANL4-3 HIV-1 60 sequence CAATTGGAGAAGTGAATTATATAAATATAAAGTAGT isolateAAAAATTGAACCATTAGGAGTAGCACCCACCAAGG GenBank:CAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGC AF324493.1 AGTGGGA Swat ATTTAAAT 61restriction site RRE AGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAG NL4-3 HIV-1 62GAAGCACTATGGGCGCAGCGTCAATGACGCTGAC isolateGGTACAGGCCAGACAATTATTGTCTGATATAGTGC GenBank:AGCAGCAGAACAATTTGCTGAGGGCTATTGAGGC AF324493.1GCAACAGCATCTGTTGCAACTCACAGTCTGGGGCA TCAAACAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCT SbfI CCTGCAGG 63 restriction site Partial envGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACC NL4-3 HIV-1 64 sequenceACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAA isolate containingATCTCTGGAACAGATTTGGAATAACATGACCTGGA GenBank: spliceTGGAGTGGGACAGAGAAATTAACAATTACACAAGC AF324493.1 acceptorTTAATACACTCCTTAATTGAAGAATCGCAAAACCAG 5A7CAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTCATAATGATA GTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAG AGTTAGGCAGGGAT ATTCACCATTATCGTTTCAGACCCACCTCCCAATC CCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCAT TCGATTAGTGAACGGATCTCGACGGT SA7AGTTAGGCAGGGATATTCACCATTATCGTTTCAGA 65 C (indicated by underlining inside env sequence above) ClaI ATCGAT 66restriction site SpeI ACTAGT 91 restriction site Partial polAGTACAAATGGCAGTATTCATCCACAATTTTAAAAG HIV-1 isolate 92 sequenceAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAA SF3 containingAGAATAGTAGACATAATAGCAACAGACATACAAACT GenBank: cPPT andAAAGAATTACAAAAACAAATTACAAAAATTCAAAATT KJ704796.1 splice TTCGGGTTTATTACAGGGACAGCAGAGATCCACT acceptor TTGG SA1 [alternatively:[NL4-3 HIV-1 93 ACAAATGGCAGTATTCATCCACAATTTTAAAAGAAA isolateAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAG GenBank:AATAGTAGACATAATAGCAACAGACATACAAACTAA AF324493.1]AGAATTACAAAAACAAATTACAAAAATTCAAAATTTT CGGGTTTATTACAGGGACAGCAGAGATCCACTTT GG ] SA1GTTTATTACAGGGACAGCAGAGATCCACTTTGG NL4-3 HIV-1 94(indicated by underlining in cPPT sequence above) isolate GenBank:AF324493.1 Blunted PstI CTGGAT 69 restriction site BclI TGATCA 70restriction site EF1alpha CGTGAGGCTCCGGTGCCCGTCAGTGGGCAGAGC GenBank: 95promoter GCACATCGCCCACAGTCCCCGAGAAGTTGGGGGG HQ644134.1AGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGG TGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGA ACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCAGAACACAGGT AAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTAC TTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAG GCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCC GCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTT TTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACT GGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAG GCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTG GTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAG TTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGC TCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCA TGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCG TCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTA GGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCA TTTCAGGTGTCGTGA ConstitutiveCAGAACACAGGTAAGTGC 72 splice donor (indicated by underlining in P-EF1a (CD) sequence above) Constitutive TCCATTTCAGGTGTCGTGA 73 splice(indicated by underlining in P-EF1a  acceptor sequence above) (CA) XbaITCTAGA 74 restriction site BamHI GGATCC 75 restriction site Kozak GCCACCPMCID: 96 sequence PMC306349 EGFP ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGGenBank: 76 TGGTGCCCATCCTGGTCGAGCTGGACGGCGACGT KJ697753.1AAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGC GAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCC CTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGA AGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGA CGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGC TGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGC CACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACA TCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTG CTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGA TCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGT AA Sall GTCGAC 77 restriction siteModified TAAACAGGCCTATTGATTGGAAAGTATGTCAACGA Hepatitis B 79 HPRE NoXATTGTGGGTCTTTTGGGGTTTGCTGCCCCTTTTAC virus isolateGCAATGTGGATATCCTGCTTTAATGCCTTTATATGC bba6,ATGTATACAAGCAAAACAGGCTTTTACTTTCTCGCC GenBank:AACTTACAAGGCCTTTCTAAGTAAACAGTATCTGAC KP341007.1CCTTTACCCCGTTGCTCGGCAACGGCCTGGTCTGT GCCAAGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCTTGGCCATAGGCCATCAGCGCATGCGTGGA ACCTTTGTGTCTCCTCTGCCGATCCATACTGCGGAACTCCTAGCCGCTTGTTTTGCTCGCAGCAGGTCTG GAGCGAAACTCATCGGGACTGACAATTCTGTCGTGCTCTCCCGCAAGTATACATCGTTTCCAGGGCTGCT AGGCTGTGCTGCCAACTGGATCCTGCGCGGGACGTCCTTTGTTTACGTCCCGTCGGCGCTGAATCCCGC GGACGACCCCTCCCGGGGCCGCTTGGGGCTCTACCGCCCGCTTCTCCGTCTGCCGTACCGACCGACCA CGGGGCGCACCTCTCTTTACGCGGACTCCCCGTCTGTGCCTTCTCATCTGCCGGACCGTGTGCACTTCG CTTCACCTCTGCACGTCGCATGGAGACCACCGTGAACGCCCACCGGAACCTGCCCAAGGTCTTGCATAA GAGGACTCTTGGACTTTCAGCAATGTCAAC(underlined codon is mutated ATG/AGG codon of X protein) EcoRI GAATTC 80restriction site SacI GAGCTC 81 restriction site KpnI GGTACC 82restriction site Partial Nef CTTTAAGACCAATGACTTACAAGGCAGCTGTAGATNL4-3 HIV-1 83 sequence, CTTAGCCACTTTTTAAAAGAAAAGGGGGG isolatecontaining GenBank: PPT AF324493.1 dU3ACTGGAAGGGCTAATTCACTCCCAACGAAGACAAG NL4-3 HIV-1 84 ATCTGCTTTTTGCTTGTACTisolate GenBank: AF324493.1 R GGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGNL4-3 HIV-1 85 AGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAG isolateCCTCAATAAAGCTTGCCTTGAGTGCTTC GenBank: AF324493.1 U5AAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGT NL4-3 HIV-1 86AACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTG isolate GAAAATCTCTAGCAG GenBank:AF324493.1 SV40 TAGTAGTTCATGTCATCTTATTATTCAGTATTTATAA GenBank: 97termination CTTGCAAAGAAATGAATATCAGAGAGTGAGAGGAA KX757255.1 andCTTGTTTATTGCAGCTTATAATGGTTACAAATAAAG polyadenylationCAATAGCATCACAAATTTCACAAATAAAGCATTTTT signalTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGCTCTAGCTATCCCG CC Kan-RATGAGCCATATTCAACGGGAAACGTCGAGGCCGC Synthesized 88GATTAAATTCCAACATGGATGCTGATTTATATGGGT by DNA2.0ATAAATGGGCTCGCGATAATGTCGGGCAATCAGGT GCGACAATCTATCGCTTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCG TTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCACTTCCGACCAT CAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCCCCGGAAAAACAGCGTTCC AGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTG CACTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGCCTCGCTCAGGCGCAATCACG AATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTC TGGAAAGAAATGCATAAACTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGT ATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTG AGTTTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGC AGTTTCATTTGATGCTCGATGAGTTTTTCTAA

Example 5. Optimization of Transfer Vectors for Lentiviral Production

An improved lentiviral transfer vector system with greater viral titers,enhanced biosafety features, improved transduction efficiency, anddurable transgene expression was developed. A number of cis-elementswere re-engineered to generate a pNLV transfer vector backbone (FIG. 6).To determine the efficacy of the GFP-encoding pNLV lentiviral transfervector compared to the GFP-encoding pCINS transfer vector, a series ofexperiments was performed, in which one of the transfer vectors wasintroduced into cells by transient transfection of packaging cells.Briefly, Expi293™ cells (Thermo Fisher) were grown in 5 ml of Freestyle(FS) medium at a concentration of 5 min/ml at 200 rpm, 3TC and 8% CO₂,80% humidity. Transfections were performed using PEIPro Transfer Reagent(PolyPlus) using 3 μg pNVS-MDLgp-RRE, 3 μg pNVS 20 RSV Rev-Kan, 0.75 μgpNVS-MDG-VSVG-Kan, and 6 μg transfer plasmid. Viral supernatants werecollected 48 hours after transfection and subjected to titer analysis(i.e., measurement of infectious titer).

The pNLV transfer vector generated a viral titer approximately two tothree times higher than that generated by the control parental (i.e.,before optimization) vector (FIG. 7 A). Physical titer was evaluatedbased on the viral RNA copy number (e.g., as measured by qRT-PCR), andthe infectious titer was determined based on the infectious unitsdetermined by proviral integration titer assay (FIG. 7 B).

Next, the level of transgene expression was examined after genomicintegration of elements from the pNLV vector compared to the pCINStransfer vector. T cells were infected with viruses produced using thepCINS or pNLV lentiviral vectors, such that viral elements wereintegrated into the T cell genomes. The pNLV vector was found to have agreater level of transgene expression (as measured by FACS) compared topCINS (FIGS. 8A-8C).

The pNLV and pCINS vectors were also compared to several commercialtransfer vectors with respect to their relative infectious titer. Eachof the commercial vectors and the pCINS vector were separately comparedto a GFP-encoding control (parental) vector, and the pCINS vector wasfurther compared to pNLV. The pLVX (Clontech) transfer vector wastransduced into 293T cells and was found to have a slightly higher viraltiter than the control vector (FIG. 9A). The pLenti6.2 (LifeTechnologies) transfer vector was transduced into cells and was found tohave a large decrease in viral titer compared to the control vector(FIG. 9B). The pD2109 (DNA2.0) transfer vector was transduced into cellsand was found to have a small decrease in viral titer compared to thecontrol vector (FIG. 9C). The pCINS transfer vector was transduced intocells and was found to have a substantially higher viral titer than thecontrol vector (FIG. 9D). The pNLV transfer vector was also transducedinto cells and was found to have a higher viral titer than the pCINSvector (FIG. 9E). These results indicate that viruses made using boththe pCINS and pNLV are, overall, significantly more infectious thanthose made using commercially available lentiviral transfer vectors,with virus made using the pNLV vector exhibiting the highest viraltiter.

To investigate the effects of splicing on infectious titer, a series ofsplice donor and splice acceptor sites were mutated for subsequent viraltiter determination (FIG. 10A). The various splice donor and spliceacceptor site mutants of the pCINS transfer vector were transfected intopackaging cells and the viral titer was compared across the panel ofmutants to a control transfer vector (FIG. 10B). All of the mutationsled to a large decrease in viral titer, indicating that the presence ofsplice sites in the transfer vector backbone is required for maximumviral titer. These observations indicate that the presence of splicesites is important for lentiviral RNA nuclear export. Transport ofgenomic lentiviral RNA from the nucleus may require interaction with thespliceosome and Rev protein. High levels of Rev protein expressed frompackaging vectors may ensure that full-size lentiviral RNA is protectedfrom splicing and transported for packaging.

Lastly, the gag and env regions of the transfer vector backbone wereremoved in a series of newly engineered transfer vector constructs todetermine the importance of the gag and env elements on infectious viraltiter. Transfer vectors with 3′ and 5′ gag and env deletions (i.e.,-gag3′, -gag5′, -env3′, and -env5′ mutants) were made (FIG. 11A) andeach vector was then transduced into separate sets of cells. The viraltiter was determined for each of the gag and/or env deletion mutants andcompared to a control lentiviral transfer vector (FIG. 11B). It wasfound that the -env3′ and -gag3′-env5′ mutants exhibited a decrease inviral titer relative to the control vector, while the -env5′ and -gag3′showed little difference in viral titer. These results indicate that the3′ gag and 5′ env regions may be altered individually with only a slighteffect on viral titer, but other gag and env truncations are detrimentalto transfer vector efficacy.

Example 6. Sequence Differences Between pNLV and pRRLSIN

The pRRLSIN transfer vector was modified to generate the pNLV vector.These nucleotide substitutions and insertions were introduced to improvethe efficacy and biosafety profile of the lentiviral transfer vector. Inone example, the pNLV psi sequence has the following sequencedifferences compared to the pRRLSIN sequence (FIG. 12A): T771C, T784G,A785G, G788A, insertion of the polynucleotide sequence “GAG” (792-794),A798C, and G924A. pNLV has a partial gag sequence beginning at position907 through position 1074. Additionally, pNLV has the following sequencedifferences in this region compared to pRRLSIN (FIG. 12B): insertion ofthe following nucleotides: A968 and A969 (resulting in a stop codon), aswell as the following substitutions: G924A, A949C, A950G, G989A, G992A,C995T, G998A, C999T, G1004A, C1007T, C1010A, T1058G, and G1064A. pNLValso has a partial env sequence beginning at position 1083 throughposition 1228. In this region, pNLV has the following sequencedifference compared to pRRLSIN (FIG. 12C): C1105A.

The pNLV RRE region has the following sequence differences compared topRRLSIN (FIG. 12D): G1291C, A1332G, and A1414G. pNLV has a region ofpartial env (containing the major splice acceptor 7 site) beginning atposition 1479 through position 1961. Additionally, pNLV has thefollowing sequence differences in this partial env region compared topRRLSIN (FIG. 12E): A1571C, T1575C, and T1866C.

pNLV has a cPPT region that begins at position 1971 through position2118, and a partial pol sequence (containing the major splice acceptor 1site) beginning at position 1974 through position 2151 (FIG. 12F). Insome instances, the cPPT region includes a sequence of 178 nucleotides.In some instances, the partial gag, partial env, and/or partial polsequences show reduced homology to wild-type viral sequences compared tothe parent vector, thereby improving the biosafety profile.Additionally, pNLV has the following sequence differences insertionswithin the cPPT and partial pol regions compared to pRRLSIN: +A1971,+ACAAATGGCAG (1974-1984), +TTCATCC (1987-1993), +A1996, +A1997, and+CGGGTTTATTACAGGGACAGCAGAGATCCACTTTGG (2116-2151).

The above-described sequence differences between pNLV and pRLLSIN areeach applicable to pCINS and pNOX, as compared to pRLLSIN, except forthe cPPT region differences. The precise nucleotide positions of thedifferences between pCINS or pNOX, relative to pRLLSIN, can beidentified by alignment of the relevant sequences provided herein.

OTHER EMBODIMENTS

All publications, patents, and patent applications mentioned in theabove specification are hereby incorporated by reference to the sameextent as if each individual publication, patent or patent applicationwas specifically and individually indicated to be incorporated byreference in its entirety. Various modifications and variations of thedescribed methods, pharmaceutical compositions, and kits of theinvention will be apparent to those skilled in the art without departingfrom the scope and spirit of the invention. Although the invention hasbeen described in connection with specific embodiments, it will beunderstood that it is capable of further modifications and that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention that are obvious to those skilled in the artare intended to be within the scope of the invention. This applicationis intended to cover any variations, uses, or adaptations of theinvention following, in general, the principles of the invention andincluding such departures from the present disclosure come within knowncustomary practice within the art to which the invention pertains andmay be applied to the essential features herein before set forth.

What is claimed is: 1-41. (canceled)
 42. A lentiviral transfer vectorcomprising a heterologous nucleic acid sequence and being characterizedby the following features: (a) comprising a promoter driving theexpression of one or more viral sequences; (b) comprising apolynucleotide encoding at least a portion of a gag protein, wherein thepolynucleotide comprises a mutated INS1 inhibitory sequence that reducesrestriction of nuclear export of RNA relative to wild-type INS1, whereinthe polynucleotide consists of 150-250 nucleotides; and (c) notcomprising an SV40 origin of replication and/or an f1 origin ofreplication.
 43. The lentiviral transfer vector of claim 42, comprisinga two nucleotide insertion that results in frame shift and prematuretermination.
 44. The lentiviral transfer vector of claim 42, furthercomprising one or more elements selected from the group consisting of apackaging signal (psi), a partial gag sequence adjacent to or partiallyoverlapping with psi, a rev-response element, a partial env sequence,and a cPPT sequence from pol.
 45. The lentiviral transfer vector ofclaim 44, wherein the cPPT sequence comprises about 150-250 nucleotidesand comprises splice acceptor SA1 sequence.
 46. The lentiviral transfervector of claim 42, further comprising one or more restriction sitespositioned between elements of said vector.
 47. The lentiviral transfervector of claim 42, further comprising a post-transcriptional regulatoryelement (PRE), which optionally is selected from: (a) a woodchuckhepatitis virus PRE (WPRE), which optionally comprises a nucleic acidsequence having at least 95% identity to SEQ ID NO: 78; and (b) ahepatitis B virus isolate bba6 PRE (HPRE), which optionally comprises anucleic acid sequence having at least 95% identity to SEQ ID NO:
 79. 48.The lentiviral transfer vector of claim 42, further comprising an EF1apromoter, wherein said EF1a promoter comprises a nucleic acid sequencehaving at least 95% identity to SEQ ID NO: 71 or SEQ ID NO:
 95. 49. Thelentiviral transfer vector of claim 42, wherein the lentiviralcomponents of said lentiviral transfer vector originate from HIV-1. 50.The lentiviral transfer vector of claim 42, wherein said heterologousnucleic acid sequence is downstream of a Kozak sequence.
 51. Thelentiviral transfer vector of claim 42, wherein the sequence encodingsaid at least a portion of said gag protein has less than 90% sequenceidentity to a corresponding region of gag protein encoded by pMDLgpRREpackaging plasmid.
 52. The lentiviral transfer vector of claim 42,wherein said lentiviral transfer vector comprises: (i) a CMV promotercomprising a nucleic acid sequence having at least 95% identity to SEQID NO: 52, (ii) an LTR R region comprising a nucleic acid sequencehaving at least 95% identity to SEQ ID NO: 53, (iii) an LTR U5 regioncomprising a nucleic acid sequence having at least 95% identity to SEQID NO: 54, (iv) a primer binding site comprising a nucleic acid sequencehaving at least 95% identity to SEQ ID NO: 55, (v) a packaging signalcomprising a nucleic acid sequence having at least 95% identity to SEQID NO: 56, (vi) a major splice donor site comprising the nucleic acidsequence of SEQ ID NO: 57, which is within said packaging signal, (vii)a partial gag sequence comprising a nucleic acid sequence having atleast 95% identity to SEQ ID NO:58, (viii) a partial env sequencecomprising a nucleic acid sequence having at least 95% identity to SEQID NO:60, (ix) a Rev-response element comprising a nucleic acid sequencehaving at least 95% identity to SEQ ID NO: 62, (x) a partial envsequence comprising a nucleic acid sequence having at least 95% identityto SEQ ID NO:64, (xi) a splice acceptor site comprising a nucleic acidsequence having at least 95% identity to SEQ ID NO: 65, which is withinsaid partial env sequence of part (x), (xii) a central polypurine tractcomprising a nucleic acid sequence having at least 95% identity to SEQID NO: 67, 92, or 93, (xiii) a splice acceptor site comprising a nucleicacid sequence having at least 95% identity to SEQ ID NO: 68 or 94, whichis within said central polypurine tract, (xiv) an EF1alpha promoterhaving at least 95% sequence identity to SEQ ID NO: 71 or 95, (xv) apolynucleotide encoding an EGFP comprising a nucleic acid sequencehaving at least 95% identity to SEQ ID NO: 76 and/or a transgenesequence, (xvi) a PRE sequence comprising a nucleic acid sequence havingat least 95% sequence identity to SEQ ID NO: 78 or 79, (xvii) a partialnef sequence comprising a nucleic acid sequence having at least 95%sequence identity to SEQ ID NO:83, (xviii) a dU3 sequence comprising anucleic acid sequencing having at least 95% sequence identity to SEQ IDNO:84, (xix) an LTR R region comprising a nucleic acid sequence havingat least 95% identity to SEQ ID NO: 85, and (xx) an LTR U5 regioncomprising a nucleic acid sequence having at least 95% identity to SEQID NO:
 86. 53. The lentiviral transfer vector of claim 42, whereinexpression of the heterologous sequence is under the control of asubgenomic promoter, which is optionally selected from the groupconsisting of a CMV promoter, an EF1 alpha promoter, and an RSVpromoter.
 54. The lentiviral transfer vector of claim 42, wherein saidheterologous nucleic acid sequence encodes a protein, wherein optionallysaid protein comprises a chimeric antigen receptor (CAR).
 55. Thelentiviral transfer vector of claim 54, wherein said CAR comprises, in aN-terminal to C-terminal direction, an antigen binding domain, atransmembrane domain, and one or more signaling domains.
 56. Thelentiviral transfer vector of claim 55, wherein said signaling domaincomprises one or more primary signaling domains, wherein optionally saidsignaling domains comprise one or more costimulatory signaling domains.57. The lentiviral transfer vector of claim 56, wherein one of said oneor more primary signaling domains comprises a CD3-zeta stimulatorydomain, wherein optionally one or more of said costimulatory signalingdomains comprises an intracellular domain selected from a costimulatoryprotein selected from the group consisting of CD27, CD28, 4-1 BB(CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, ICAM-1, lymphocytefunction-associated antigen-1 (LFA-1), CD2, CDS, CD7, CD287, LIGHT,NKG2C, NKG2D, SLAMF7, NKp80, NKp30, NKp44, NKp46, CD160, B7-H3, and aligand that specifically binds with CD83.
 58. The lentiviral transfervector of claim 57, wherein said one or more of said costimulatorysignaling domains comprises the 4-1 BB (CD137) costimulatory domain,wherein optionally one or more of said costimulatory domains comprisesthe CD28 costimulatory domain.
 59. The lentiviral transfer vector ofclaim 55, wherein said antigen binding domain is an scFv.
 60. Thelentiviral transfer vector of claim 55, wherein said antigen bindingdomain binds to an antigen selected from the group consisting of CD19;CD123; CD22; CD30; CD171; CS-1; C-type lectin-like molecule-1, CD33;epidermal growth factor receptor variant III (EGFRvIII); ganglioside G2(GD2); ganglioside GD3; TNF receptor family member B cell maturation(BCMA); Tn antigen ((Tn Ag) or (GaINAcα-Ser/Thr)); prostate-specificmembrane antigen (PSMA); Receptor tyrosine kinase-like orphan receptor 1(ROR1); Fms-Like Tyrosine Kinase 3 (FLT3); Tumor-associated glycoprotein72 (TAG72); CD38; CD44v6; Carcinoembryonic antigen (CEA); Epithelialcell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117);Interleukin-13 receptor subunit alpha-2; mesothelin; Interleukin 11receptor alpha (IL-11 Ra); prostate stem cell antigen (PSCA); ProteaseSerine 21; vascular endothelial growth factor receptor 2 (VEGFR2);Lewis(Y) antigen; CD24; Platelet-derived growth factor receptor beta(PDGFR-beta); Stage-specific embryonic antigen-4 (SSEA-4); CD20; Folatereceptor alpha; Receptor tyrosine-protein kinase ERBB2 (Her2/neu); Mucin1, cell surface associated (MUC1); epidermal growth factor receptor(EGFR); neural cell adhesion molecule (NCAM); Prostase; prostatic acidphosphatase (PAP); elongation factor 2 mutated (ELF2M); Ephrin B2;fibroblast activation protein alpha (FAP); insulin-like growth factor 1receptor (IGF-I receptor), carbonic anhydrase IX (CAIX); Proteasome(Prosome, Macropain) Subunit, Beta Type, 9 (LMP2); glycoprotein 100(gp100); oncogene fusion protein consisting of breakpoint cluster region(BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl)(bcr-abl); tyrosinase; ephrin type-A receptor 2 (EphA2); Fucosyl GM1;sialyl Lewis adhesion molecule (sLe); ganglioside GM3; transglutaminase5 (TGS5); high molecular weight-melanoma-associated antigen (HMWMAA);o-acetyl-GD2 ganglioside (OAcGD2); Folate receptor beta; tumorendothelial marker 1 (TEM1/CD248); tumor endothelial marker 7-related(TEM7R); claudin 6 (CLDN6); thyroid stimulating hormone receptor (TSHR);G protein-coupled receptor class C group 5, member D (GPRC5D);chromosome X open reading frame 61 (CXORF61); CD97; CD179a; anaplasticlymphoma kinase (ALK); Polysialic acid; placenta-specific 1 (PLAC1);hexasaccharide portion of globoH glycoceramide (GloboH); mammary glanddifferentiation antigen (NY-BR-1); uroplakin 2 (UPK2); Hepatitis A viruscellular receptor 1 (HAVCR1); adrenoceptor beta 3 (ADRB3); pannexin 3(PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6complex, locus K 9 (LY6K); Olfactory receptor 51E2 (OR51E2); TCR GammaAlternate Reading Frame Protein (TARP); Wilms tumor protein (WT1);Cancer/testis antigen 1 (NY-ESO-1); Cancer/testis antigen 2 (LAGE-1a);Melanoma-associated antigen 1 (MAGE-A1); ETS translocation-variant gene6, located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); XAntigen Family, Member 1A (XAGE1); angiopoietin-binding cell surfacereceptor 2 (Tie 2); melanoma cancer testis antigen-1 (MAD-CT-1);melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1;tumor protein p53 (p53); p53 mutant; prostein; surviving; telomerase;prostate carcinoma tumor antigen-1, melanoma antigen recognized by Tcells 1; Rat sarcoma (Ras) mutant; human Telomerase reversetranscriptase (hTERT); sarcoma translocation breakpoints; melanomainhibitor of apoptosis (ML-IAP); ERG (transmembrane protease, serine 2(TMPRSS2) ETS fusion gene); N-Acetyl glucosaminyl-transferase V (NA17);paired box protein Pax-3 (PAX3); Androgen receptor; Cyclin B1; v-mycavian myelocytomatosis viral oncogene neuroblastoma derived homolog(MYCN); Ras Homolog Family Member C (RhoC); Tyrosinase-related protein 2(TRP-2); Cytochrome P450 1 B1 (CYP1B1); CCCTC-Binding Factor (ZincFinger Protein)-Like, Squamous Cell Carcinoma Antigen Recognized By TCells 3 (SART3); Paired box protein Pax-5 (PAX5); proacrosin bindingprotein sp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase(LCK); A kinase anchor protein 4 (AKAP-4); synovial sarcoma, Xbreakpoint 2 (SSX2); Receptor for Advanced Glycation Endproducts(RAGE-1); renal ubiquitous 1 (RU1); renal ubiquitous 2 (RU2); legumain;human papilloma virus E6 (HPV E6); human papilloma virus E7 (HPV E7);intestinal carboxyl esterase; heat shock protein 70-2 mutated (muthsp70-2); CD79a; CD79b; CD72; Leukocyte-associated immunoglobulin-likereceptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR or CD89);Leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2);CD300 molecule-like family member f (CD300LF); C-type lectin domainfamily 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2);EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2);lymphocyte antigen 75 (LY75); Glypican-3 (GPC3); Fc receptor-like 5(FCRL5); and immunoglobulin lambda-like polypeptide 1 (IGLL1).
 61. Thelentiviral transfer vector of claim 60, wherein said antigen bindingdomain binds to CD19, mesothelin, or CD123.
 62. The lentiviral transfervector of claim 54, wherein said CAR comprises an anti-CD19 antibody ora fragment thereof, a 4-1 BB (CD137) transmembrane domain, and aCD3-zeta signaling domain.
 63. A lentiviral transfer vector comprising,from 5′ to 3′, one or more of the following elements in operableassociation: (a) a promoter to drive the expression of viral genes, (b)a packaging signal (psi) comprising a major splice donor site (SD), (c)a partial gag sequence, (d) a partial env sequence, (e) a Rev-responseelement (RRE), (f) a partial env sequence comprising splice acceptorsite (SA7), (g) a central polypurine tract (cPPT) comprising a spliceacceptor site (SA1), (h) a subgenomic promoter, (i) a gene encoding EGFPand/or a heterologous nucleic acid sequence, and (j) apost-transcriptional regulatory element, which optionally comprises awoodchuck hepatitis virus PRE (WPRE) or a hepatitis B virus isolate bba6PRE (HPRE).
 64. A lentiviral transfer vector of claim 63 comprising,from 5′ to 3′, one or more of the following elements in operableassociation: (a) a CMV promoter, (b) an LTR R region, (c) an LTR U5region, (d) a primer binding site (PBS), (e) a packaging signal (psi)comprising a major splice donor site (SD), (f) a partial gag sequence,(g) a partial env sequence, (h) a Rev-response element (RRE), (i) apartial env sequence comprising splice acceptor site (SA7), (j) acentral polypurine tract (cPPT) comprising a splice acceptor site (SA1),(k) an EF1a promoter, (l) a gene encoding EGFP and/or a heterologousnucleic acid sequence, (m) a post-transcriptional regulatory element,which optionally comprises a woodchuck hepatitis virus PRE (WPRE) or ahepatitis B virus isolate bba6 PRE (HPRE), (n) an LTR R region, (o) anLTR U5 region, (p) an SV40 polyA tail, (q) a kanamycin resistance gene(nptII), and (r) a pUC origin of replication.
 65. The lentiviraltransfer vector of claim 63, wherein said heterologous nucleic acidsequence encodes a chimeric antigen receptor (CAR), which optionallycomprises an anti-CD19 antibody or a fragment thereof, a 4-1 BB (CD137)transmembrane domain, and a CD3-zeta signaling domain.
 66. A host cellcomprising the lentiviral transfer vector of claim 42, whereinoptionally: (a) said host cell is a 293T cell, a Jurkat T cell, or aprimary human T cell; and/or (b) said host cell further comprises one ormore lentiviral packaging vectors.
 67. A composition comprising alentiviral transfer vector of claim 42 and one or more packagingvectors.
 68. A method of producing a lentivirus capable of expressing aheterologous nucleic acid sequence, said method comprising: (a)introducing into a cell: (i) the lentiviral transfer vector of claim 42,and (ii) one or more lentiviral packaging vectors; and (b) expressingviral proteins encoded by said lentiviral transfer vector and/or saidpackaging vector in said cell, thereby producing a lentivirus comprisingthe heterologous nucleic acid sequence of said lentiviral transfervector, wherein optionally said cell is a 293T cell, a Jurkat T cell, ora primary human T cell.